Analyte sensors, methods for preparing and using such sensors, and methods of detecting analyte activity

ABSTRACT

Analyte sensors, methods for producing and using analyte sensors, methods of detecting and/or measuring analyte activity, detecting pH change, and/or, controlling the concentration of an analyte in a system, are disclosed. Embodiments of the analyte sensors according to the disclosure can provide an accurate and convenient method for characterizing analyte activity, detecting pH change, controlling the concentration of an analyte in a system, and the like, in both in vivo and in vitro environments, in particular in living cell imaging.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/394,501, entitled “ANALYTE SENSORS, METHODS FOR PREPARINGAND USING SUCH SENSORS, AND METHODS OF DETECTING ANALYTE ACTIVITY” filedon Oct. 19, 2010, the entirety of which is hereby incorporated byreference. This application also claims priority to U.S. ProvisionalPatent Application Ser. No. 61/526,420, entitled “ANALYTE SENSORS,METHODS FOR PREPARING AND USING SUCH SENSORS, AND METHODS OF DETECTINGANALYTE ACTIVITY” filed on Aug. 23, 2011, the entirety of which ishereby incorporated by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to fusion protein analyte sensorscomprising an analyte binding region and a fluorescent polypeptide forthe detection of metal ion analytes and to methods of their use in vivoand in vitro.

SEQUENCE LISTING

The present disclosure includes a sequence listing incorporated hereinby reference in its entirety.

BACKGROUND

Ca²⁺ is the most ubiquitous signaling molecule in the human body,regulating numerous biological functions that include heart beat, musclecontraction, neural function, cell development, and proliferation, byfluxing between the subcellular compartments with different amplitudesand durations[1]. The membrane-based organelle endo/sarcoplasmicreticulum (ER/SR) lumen, which occupies less than 10% of cell volume,stores more than 90% of intracellular Ca²⁺ and is pivotal in controllingCa²⁺ signaling. It can produce intrinsic Ca²⁺ release and propagation ofCa²⁺ oscillations[2-4]. Ca²⁺-mobilization agonists such as ATP,ionomycin, histamine, and glutamine will activate Ca²⁺ receptors andpumps, such as inositol 1,4,5-trisphosphate receptor (IP₃R), to releaseCa²⁺ from the ER into the cytosol[5-7], which results in a rapiddecrease of ER Ca²⁺ (from mM at the resting state to μM in excitedstate). The removal of these agonists will help Ca²⁺ refill the ERthrough membrane channels such as sarco(endo)plasmic reticulumCa²⁺-ATPase (SERCA). The alternation of Ca²⁺ concentration activatesvarious intracellular Ca²⁺ sensing (trigger) proteins, such ascalmodulin (CaM), troponin C (TnC) and other ion channels, throughconformational changes that occur upon binding to Ca²⁺[8]. Theseactivated Ca²⁺-sensor receptors will further regulate numerous cellularprocesses and events. Recent studies indicate that Ca²⁺ signaling isimportant for homeostatic handling of cardiovascular functions[9-11]. Incardiomyocytes, cardiac relaxation and contraction is regulated by theperiodic change of intracellular Ca²⁺ concentration and the proteinsassociated with the sarcoplasmic reticulum (SR), a homologue of ER[12,13]. The cardiac ryanodine receptor (RyR2), inositol(1,4,5)-trisphosphate receptor (IP₃R) and the sarcoplasmic reticulumCa²⁺-ATPase 2a (SERCA2a) are three pivotal portals for the Ca²⁺mobilization during this agonist-induced process. Heart failure causedby dysfunction of these two proteins, associated with abnormal Ca²⁺handling, is becoming increasingly evident in data collected both fromanimals and humans[14-17]. A Ca²⁺ indicator to monitor ER/SR Ca²⁺concentrations with fast release kinetics, and the capability toquantitatively detect Ca²⁺ signaling in specific subcellular organelleswill have a significant impact on the understanding of the molecularbasis of Ca²⁺ signaling and homeostasis in cardiac development anddiseases.

The initial measure of ER Ca²⁺ dynamics was achieved using the Ca²⁺ dyeMag-fura-2 in plasma membrane-permeabilized live cells. In contrast toCa²⁺ dyes, fluorescent protein (FP)-based Ca²⁺ indicators withgenetically encoded chromophores can detect Ca²⁺ signaling insubcellular organelles with high spatial and temporal resolution. Theyconsist of a Ca²⁺-modulated protein, either calmodulin or troponin C,coupled to a single fluorescent protein to generate sensors, such asGCaMP (11), or dual fluorescent proteins, such as Cameleon. ModifyingCameleon at its Ca²⁺ binding loops or CaM's peptide-interaction surfacegenerated several ER/SR sensors, which have been applied to excitablecells with some limitations. Directly monitoring fast ER/SR Ca²⁺dynamics in excitable cells is still new territory.

As a secondary messenger, calcium ions regulate many biologicalprocesses in various intracellular compartments through interactionswith proteins. Calcium is involved in muscle contraction (includingheartbeat), vision, and neuronal signaling. Calcium binding proteinsexhibit different calcium binding affinities with K_(d) ranging from 0.1μM to mM, which are essential for their responses to various stimulithrough the temporal and spatial changes of calcium and calciumhomeostasis. For example, extracellular calcium-modulated proteins withmultiple calcium binding sites, such as cadherins and calcium-sensingreceptors, have dissociation constants in the submillimolar tomillimolar range. Calsequestrin, a major calcium binding protein in theendoplasmic reticulum (ER), has a relatively weak calcium bindingaffinity that enables it to release or bind calcium in the ER calciumstore.

The endoplasmic reticulum (ER) with a resting Ca²⁺ concentrationfunctions as the primary intracellular Ca²⁺ store, which can produceboth a synchronous Ca²⁺ release and propagating Ca²⁺ waves.Ca²⁺-mobilizing agonists such as ATP, histamine, and glutamine, andsecond messengers, such as IP₃ and cADPR, generate an increase in thecytosolic Ca²⁺ concentration ([Ca²⁺]_(c)) with a defined spatio-temporalpattern. The release of Ca²⁺ from the ER stores results in a rapidincrease in [Ca²⁺]_(c) (from approximately 10⁻⁷ M at the resting stateto approximately 10⁻⁶ M in the excited state) that activates a number ofintracellular Ca²⁺ sensing (trigger) proteins including calmodulin(CaM), troponin C (TnC), and other ion channels and enzymes (ProteinSci. 7: 270-282). While the prevalence of calcium throughout thebiological system is well-known and extensive efforts have been made,understanding the calcium regulation of biological functions, stability,folding, and dynamic properties of proteins is limited largely due tothe calcium-dependent conformational changes and cooperative calciumbinding in natural proteins.

The study of the key determinants of calcium binding has been acontinuing endeavor for decades. There are several factors, such as thetype, charge, and arrangement of the calcium ligands that have beenshown to be important in calcium binding. Calcium is mainly chelated bythe oxygen atoms from the sidechains of Asp, Asn, and Glu, themain-chain carbonyl, and solvent water molecules in proteins; thepentagonal bipyramid geometry is the most popular binding geometry.Because of the electrostatic nature of calcium binding, charged Asp andGlu occur most often in calcium binding sites. The charge number in thecoordination sphere also plays a role in calcium binding affinity. Inaddition, a more electronegative environment causes a stronger bindingaffinity for a given calcium site, and the electrostatic environmentaffects the cooperativity in multi-site systems. For these multi-siteproteins, the apparent calcium affinity contains contributions from themetal-metal interactions and the cooperativity of the binding sites.However, quantitative estimation of the key factors for calcium bindingis yet to be established. Therefore, the systematic study of the keydeterminants for calcium binding required a new strategy and modelsystem.

Monitoring the effects of calcium on the abundant cellular processeshas, thus far, been a difficult endeavor due to numerous factors, suchas interference from endogenous proteins and perturbation of originalcalcium signal pathways. While commercially available dyes with bindingaffinities ranging from 60 nM to hundreds of micromolar can be loadedinto mammalian cells through simple incubation, they cannot be targetedto specific cell compartments in a predictable amount, causingdifficulty in accurately determining the dye concentration andmonitoring calcium concentration. Many of these dyes were shown to havebuffering effect in cells and do not provide the necessary sensitivityfor thick tissues, intact organisms, or non-mammalian cells.Protein-based calcium sensors that can be directly expressed by thecells and reliably targeted to specific subcompartments have been usedin a wide variety of cell types, including mammalian and bacteria.Aequorin was first applied to monitor calcium responses at differentcellular environments. However, aequorin requires the constant additionof coelenterazine, which is consumed after each reaction.

FRET-based calcium sensors were then developed using two differentlycolored fluorescent proteins or their variants linked with a calmdoulinbinding peptide and calmodulin (Cell Calcium 22: 209-216; Nature, 388:882-887). To avoid using the essential trigger protein calmodulin,Troponin C (TnC) was used to sense calcium concentration change in theFRET pair of fluorescent proteins. To address the major concernregarding the competition of endogenous protein and the perturbation ofthe natural calcium signal systems using essential proteins such ascalmodulin and troponin C and the potential perturbation of the naturalcalcium signal network, a modification of calmodulin binding sites andcalmodulin to reduce the interaction was performed (Proc. Natl. Acad.Sci. U.S.A. 101: 17404-17409; Chem. Biol. 13: 521-530). Therefore, thereremains a need to develop calcium sensors without using natural calciumbinding proteins to monitor the spatial and temporal changes of calciumin the cell, especially at high concentration organelles such as theendoplasmic reticulum.

Endoplasmic reticulum/Sarcoplasmic reticulum calcium signaling arecrucial for the research of muscle contraction, brain activity and allthe other calcium mishandling related diseases. Different from bulkvolume of cytosol in cells, ER/SR has well defined outline and onlytakes 3% of the total volume of the cell, which is challenged to bestudies without highly specific-target calcium indicators.Unfortunately, there are only a few genetically encoded ER calciumsensor published, and all the Kds narrowed around tens of micromolar,while it is well known that free calcium concentration in SR of skeletalmuscle cell is around 1 mM, with extra 20 mM calcium bound bycalsequestrin. There is a strong need to design an SR calcium sensorwith lower binding affinity which is appropriated to measure SR calciumin the muscle cells or tissues. Ideally, the calcium binding affinityshould be around 1 mM or sub-millimolar range, similar to the overallcalcium binding affinity of SR calcium buffer protein calsequestrin,which is based on the strategy that the cytosolic calcium indicatorssuch as fura-2, camelone and GcamP2 and so on exhibit Kd aroundsub-micromolar, within the same magnitude of Kd of calmodulin.

The fluorescence change of calmodulin-based calcium sensors highlyrelies on the interaction between calcium bound form calmodulin and M13peptide, which is a bulk complex with several different bindingprocesses. The calcium binding affinities to C- and N-domain ofcalmodulin are in different magnitudes. Moreover, holo-form calmodulinand M13 peptide interact will add an additional Kd to the overallbinding process, so the apparent Kd of the sensors does not directlycome from the calcium binding, but in a mixture of two Kds withdifferent magnitudes from calcium and calmodulin interaction and asequential Kd from the calmodulin and M13 peptide interaction. Thecalmodulin based calcium indicator cannot quantitatively measure thecalcium change, as the equation of D1ER binding process involvingseveral constants such as Kd1, Kd2 and Hill coefficients which aredifficult to be measured in situ. Furthermore, the kinetics of CaM andM13 peptide interaction could not be further accelerated due to complexdelay.

SUMMARY

Embodiments of the present methodology provides designing Ca²⁺ biosensorby creating a Ca²⁺ binding site on GFP with site-direct mutagenesis,which not only overcomes the limitations of current Ca²⁺ sensors, butalso can be utilized in various other fluorescent proteins withdifferent optical properties for the further application in tissue andanimal imaging, to accurately measure the real-time Ca²⁺ concentrationin ER, which enhances our understanding of Ca²⁺ signaling in ER,correlated to its biological function. Embodiments of the disclosureprovides enhanced sensors with different signal peptides andmultiple-magnitude binding affinities, which can help in detecting Ca²⁺signaling response to different agonists in various subcellularorganelles of diverse cell types.

One aspect of the disclosure, therefore, encompasses embodiments of anengineered fluorescent host polypeptide having a metal ion binding sitecomprising a plurality of negatively charged residues, wherein thenegatively charged residues comprise a plurality of carboxyl oxygensorientated in a pentagonal bipyrimdal geometry wherein said geometryprovides a metallic ion binding site operatively interacting with achromophore region of the engineered fluorescent host polypeptide suchthat binding of a metal ion analyte to the molecular recognition motifmodulates the emission of a fluorescent signal emitted by thefluorescent host polypeptide, and optionally, the absorbance spectrum ofthe engineered fluorescent host polypeptide.

Another aspect of the disclosure encompasses embodiments of acomposition comprising an embodiment of the analyte sensor, where thecomposition can be formulated for the detection of an analyte in a testsample.

Yet another aspect of the disclosure encompasses embodiments of a kitcomprising an analyte sensor according to the disclosure and packaging,the packing comprising instructions for the use of the analyte sensorfor the detection of an analyte by the analyte sensor.

Still another aspect of the disclosure encompasses embodiments of amethod for detecting an analyte, comprising: (i) providing an analytesensor according to the disclosure; (ii) providing a test samplesuspected of comprising an analyte having affinity for the molecularrecognition motif of the analyte sensor; (iii) detecting a firstfluorescent signal emitted by the analyte sensor in the absence of atest sample suspected of comprising an analyte having affinity for themolecular recognition motif of the analyte sensor; (iv) contacting theanalyte sensor with the test sample; (v) detecting a second fluorescentsignal emitted by the analyte sensor in contact with the test sample;and (vi) comparing the first fluorescent signal and the secondfluorescent signal, wherein a ratiometric change in the signal indicatesan analyte in the test sample is interacting with the analyte sensor.

Another aspect of the disclosure encompasses embodiments of arecombinant nucleic acid encoding an analyte sensor according to thedisclosure.

Another aspect of the disclosure encompasses embodiments of a method forcharacterizing the cellular activity of an analyte comprising: (i)providing a genetically modified cell comprising a recombinant nucleicacid expressing an analyte sensor according to claim 1; (ii) expressingthe analyte sensor in the genetically modifying a cell measuring asignal produced from the analyte sensor; (iii) detecting a firstfluorescent signal emitted by the analyte sensor; (iv) detecting asecond fluorescent signal emitted by the analyte sensor after theinduction of a physiological event in the cell; and (v) comparing thefirst fluorescent signal and the second fluorescent signal, wherein aratiometric change in the signal indicates a change in the level of theanalyte in the cell associated with the physiological in cell.

BRIEF DESCRIPTION OF THE FIGURES

Further aspects of the present disclosure will be more readilyappreciated upon review of the detailed description of its variousembodiments, described below, when taken in conjunction with theaccompanying drawings.

FIGS. 1A and 1B illustrate a model structure of EGFP-based Ca²⁺ sensorsbased on 1ema.pdb. All Ca²⁺ sensors were composed of a Ca²⁺ bindingmotif integrated into an enhanced green fluorescent protein (EGFP).

FIG. 1A illustrates the domain structures of various GFP variants.CRsig: the calreticulin signal peptide MLLSVPLLLGLLGLAAAD (SEQ ID No.:112); KDEL: ER retention signal; kz: Kozak consensus sequence foroptimal translational initiation in mammalian cells. Constructs Ca-G1and Ca-G2 contain the flanking sequences. Ca-G1′, Ca-G2′ and Ca-G3′ donot contain flanking sequences.

FIG. 1B schematically illustrates the topology of Glu172-Asp173(position 1), Gln157-Lys158 (position 2), and Asn144-Tyr145 (position 3)in EGFP.

FIG. 2A illustrates the visible absorbances of EGFP-wt and variantsthereof. Protein concentrations were 20 μM.

FIG. 2B illustrates the fluorescence spectra of EGFP-wt and variantsthereof. Protein concentrations were 10 μM; slit width of 1 nm for bothexcitation and emission. λ_(ex)=398 nm.

FIGS. 3A-3D illustrate the spectroscopic characterizations of the Ca²⁺sensor Ca-G1-37.

FIG. 3A illustrates the visible absorption spectra for sensor Ca-G1-37at 17 μM at various Ca²⁺ concentrations. Arrows indicate the directionof signal change resulting from an increase in the Ca²⁺ concentration.

FIG. 3B illustrates the Ca²⁺ dependence of fluorescence emission spectrawith excitation of λ_(ex)=398 nm at 1.7 μM at various Ca²⁺concentrations. Slit width of excitation and emission was 1 and 2 nm,respectively. Arrows indicate the direction of signal change resultingfrom an increase in the Ca²⁺ concentration.

FIG. 3C illustrates the Ca²⁺ dependence of fluorescence emission spectrawith excitation of λ_(ex)=490 nm at 1.7 μM at various Ca²⁺concentrations. Slit width of excitation and emission was 1 and 2 nm,respectively. Arrows indicate the direction of signal change resultingfrom an increase in the Ca²⁺ concentration.

FIG. 3D is a graph showing normalized F_((398nm))/F_((490nm)) ratiocurve-fitting of Ca²⁺ titration data.

FIG. 4A illustrates Ca²⁺ responses of Ca-G1-37 in the presence of: Cu²⁺(0.1 μM), Zn²⁺ (0.1 mM), Mg²⁺ (10.0 mM), Tb³⁺ (5.0 μM) and La³⁺ (5.0μM). The ratio of fluorescence emission of Ca-G1-37 with 398 nm and 490nm excitation in the presence of 1.0 mM Ca²⁺ was used to normalize thevalues using Eq. (5).

FIG. 4B illustrates Ca²⁺ responses of Ca-G1-37 in the presence of: theintracellular molecules: ATP (0.2 mM), ADP (0.2 mM), GTP (0.1 mM), GDP(0.1 mM), and GSH (1.0 mM). The ratio of fluorescence emission ofCa-G1-37 with 398 nm and 490 nm excitation in the presence of 1.0 mMCa²⁺ was used to normalize the values using Eq. (5).

FIGS. 5A-5D illustrate a kinetic analysis of Ca²⁺ association to Ca-G1.FIG. 5A: Stopped-flow traces of fluorescence increase (λ_(ex)=398 nm)upon rapid mixing of Ca-G1 (final concentration of 20 μM) and Ca²⁺ atconcentrations indicated. FIG. 5B: Observed rates of fluorescenceincreases as a function of Ca²⁺ concentration. FIG. 5C: Maximal changesin the amplitude of the fluorescence intensities observed in FIG. 5A asa function of Ca²⁺ concentration. FIG. 5D: Stopped-flow trace offluorescence decrease (λ_(ex)=398 nm) upon rapid mixing of 40 μM Ca-G1preloaded with 0.8 mM Ca²⁺. A 455 nm long pass filter was used tocollect the emission with a main peak at 510 nm. Data were fit to Eq. 6(FIGS. 5A and 5D), Eq. 8 (FIG. 5B) and Eq. 2 (FIG. 5C), respectively.

FIGS. 6A-6D is a series of digital images illustrating the localizationof sensor Ca-G1-ER in HeLa and BHK-21 cells. FIG. 6A: Localization ofCa-G1-ER; FIG. 6B: Localization of DsRed2-ER; FIG. 6C: overlay ofCa-G1-ER and DsRed2-ER in HeLa cells; FIG. 6D: Localization of Ca-G1-ERin BHK-21 cell. Confocal images of Ca-G1-ER and DsRed2-ER localizationwere with an argon laser 488 nm line for the green channel, and a He—Nelaser 543 nm line for the red channel. The scale bar indicates 10 μm.

FIGS. 6E and 6F illustrate the calcium response of the sensor Ca-G1-ERin BHK-21 cells. FIG. 6E: Time course of Ca²⁺ responses in response todifferent treatments. FIG. 6F: pseudo calibration of Ca²⁺ concentrationsin the ER. The time course expressed as the fluorescence emission ratioat 510 nm for excitation at 385 and 480 nm. The left-hand ordinaterepresents the 510 nm fluorescence emission ratio (excitation 385 and480 nm) in both FIGS. 6E and 6F; right-hand ordinate represents thecalibrated Ca²⁺ concentration in the ER in FIG. 6F.

FIG. 7 illustrates CD spectra of EGFP-wt and variants thereof in 10 mMTris and 1 mM DTT (pH 7.4). The protein concentrations were 10 μM for CDexperiments.

FIG. 8A illustrates visible absorbance spectra of Ca-G1′ at various pHs.Measurements were performed in 1 mM DTT and 10 mM MES (pH 5.0, 5.5,6.0), 10 mM PIPES (pH 6.5, 7.0), and 10 mM Tris (pH 7.4, 8.0, 9.0).

FIG. 8B illustrates the curve fitting of Ca-G1′ at various pHs.

FIG. 9A illustrates a model structure of calcium binding fluorescentprotein with the addition of the EGFP.D2 (site 1) by computationaldesign or EGFP-G1 (172EF) by inserting the EF-hand motif III fromcalmodulin into position 172-173. Residues involved in the formation ofthe chromophore are highlighted. The structure of EGFP around thechromophore based on 1EMA.pdb.

FIG. 9B illustrates a model structure of modified grafting EGFP sensor.One EF-hand was inserted in the fluorescent sensitive location of EGFP,generating EGFP-G1. A site-directed mutagenesis on the beta-sheetsurface introducing a negatively charged residue to form a Ca²⁺ bindingsite with three existed negatively charged residues.

FIG. 10 is a graph illustrating expression of EGFP and variants thereofin E. coli BL21-DE3 22 hrs after 200 mM IPTG induction and at 30° C.(open bars) and 37° C. (closed bars), respectively. λex=488 nm.

FIG. 11 is a series of digital images illustrating fluorescencemicroscope imaging of HeLa cells. The imaging was performed two days (48hrs) after HeLa cells were transfected with EGFP-G1, EGFP-G1-C2, andEGFP-G1-C3. The exposure time was 200 ms.

FIG. 12A is a graph illustrating EGFP-D2 series expression in HeLa cellsat 30° C. and 37° C., respectively. Fluorescence intensity at 510 nm ofdifferent cell pellets was obtained for 2 days after transfection of theproteins. λ_(ex)=488 nm.

FIG. 12B is a graph illustrating EGFP-G1 series expression in HeLa cellsat 30° C. and 37° C., respectively. Fluorescence intensity at 510 nm ofdifferent cell pellets was obtained for 2 days after transfection of theproteins. λ_(ex)=488 nm.

FIG. 13A illustrates the visible absorbance spectra of EGFP, EF-172, andSite 1. The protein concentrations were 2 mM.

FIG. 13B illustrates the fluorescence spectra of EGFP, EF-172, andSite 1. The protein concentrations were 2 mM. Slit width of 1 nm forboth excitation and emission; λ_(ex)=488 nm.

FIG. 14 schematically illustrates a calcium-binding protein based on GFP(pdb 1b9c). The binding geometry of GFP.D1 is shown in ball-and-stick.D2 is shown as a circle. The locations of GFP.D3 with the wild typeresidues are also indicted.

FIG. 15A is a graph illustrating the absorbance spectra of EGFP, GFP.D1,GFP.D2, GFP.D2′ and GFP.D2″ expressed in E. coli, indicate that thechromophore of GFP.D1 did not form.

FIG. 15B illustrates the far UV CD spectra of EGFP, GFP.D1, GFP.D2,GFP.D2′ and GFP.D2″ indicating the formation of β-sheet secondarystructures with a negative maximum at 216 nm.

FIG. 16 is a series of digital images illustrating the invertedepifluorescence image of HeLa cells expressing: (a) wild-type EGFP; (b)GFP.D1; and (c) GFP.D2.

FIG. 17A illustrates the calcium-induced chromophore emission change forGFP.D2 expressed in E. coli with excitation at 482 nm. The fit of thedata using the 1:1 binding equation (Eq. 2.3) gives a K_(d) of 107 μM.

FIG. 17B illustrates the rhodamine-5N competition with GFP.D2 forcalcium binding fluorescence emission with excitation at 552 nm. Theinset shows the spectra of the Rhodamine-5N with the concentrationchange of Ca²⁺.

FIG. 17C illustrates the fluorescence change of 3 μM GFP.D1 in 20 mMPIPES, 10 mM KCl, 1 mM DTT, 1% glycerol, pH 6.8 at increasing terbiumconcentrations assuming a 1:1 binding. The inset shows the spectra peaksincrease at 545 nm.

FIG. 17D illustrates the metal competition of GFP.D1.

FIG. 18 illustrates competitive titration of Pb²⁺ and Ca²⁺-loaded EGFPvariants C2 and C4.

FIG. 19A illustrates titration of excess Pb²⁺ with Ca²⁺-loaded EGFPvariant C2. Signal intensity decreases as Pb displaces Ca.

FIG. 19B illustrates the curve-fitting of C2-Pb complex to quantify Kd.The Kd for C2-Pb²⁺ was 2 μM, and Ca²⁺ was 440 μM.

FIG. 19C illustrates curve fitting of EGFP/Pb²⁺ complex to quantify Kd.The Kd was 3.5 μM.

FIG. 19D illustrates curve-fitting of C2-Gd complex to quantify Kd. TheKd for Gd³⁺ was 2.0 μM.

FIGS. 20A-20L illustrate the structure and in vitro optical propertiesof Ca²⁺ biosensor variants.

FIG. 20A schematically illustrates a truncated structure (left image) ofthe wild-type EGFP (1EMA) with the chromophore (CRO) shown as spheres.Residues 147, 202, 204, 223, and 225 sidechain protruding from thesurface in close proximity to the chromophore were mutated to form theCa²⁺ binding ligands. Key residues H147, T203, and E222, involved inproton interaction with the chromophore are located near the designedCa²⁺ binding site.

FIG. 20B illustrates the spatial distribution of the five residues thatare responsible for Ca²⁺ chelation.

FIG. 20C illustrates the spatial organization of the five residuesindicated in FIG. 20B and their relationship with the chromophore in theEGFP molecule, which shows nonacidic residues.

FIGS. 20D-20H, respectively illustrate constructs D8, D9, D10, CatchER,and D12 and show replacement at residues S147, S202, Q204, F223, andT225, respectively.

FIG. 20I illustrates the absorbance spectra of wild-type EGFP and theCa²⁺ sensors D8 to D12, with a normalized absorbance peak at 280 nm. TheCa²⁺ sensors D8 to D12 exhibited a major absorbance peak at 398 nm and alower peak at 490 nm.

FIG. 20J illustrates the absorbance intensity ratio at 395 nm and 488 nmfor the Ca²⁺ sensors D8 to D12 and wild-type EGFP. The ratio increasedwith the number of negatively charged residues introduced.

FIG. 20K illustrates the change in fluorescence intensity of EGFPvariants in response to Ca²⁺ recorded at 510 nm emission and 488/395 nmexcitation with either 10 μM EGTA (black/grey bars) or 5 mM Ca²⁺(red/blue bars). EGFP emission maxima at 510 nm, excited at 488 nm, inthe presence of 10 μM EGTA were normalized to 1.0.

FIG. 20L illustrates the correlation between the number of negativelycharged residues and apparent Ca²⁺ dissociation constants (K_(d)) forD9, D10, and CatchER, measured by fluorescence titration in the presence(square) and absence (circle) of 100 mM KCl.

FIGS. 21A-21E illustrate the optical characterizations of CatchER invitro.

FIG. 21A shows emission spectra in response to increased Ca²⁺concentrations.

FIG. 21B shows the CatchER apparent K_(d) determined by fluorescenceresponse in the presence or absence of 100 mM KCl, or by a main chainchemical shift change of residue Y143 in heteronuclear single quantumcoherence (HSQC) spectra in the presence of 10 mM KCl (black). Titrationresults were fitted to a 1:1 binding mode.

FIG. 21C shows the fluorescence responses of various physiologicalmolecules: 20 mM Na⁺, 100 mM K⁴, 2 μM Cu²⁺, 2 μM Zn²⁺, 1 mM Mg²⁺, 0.2 mMATP, 0.1 mM GTP, and 0.1 mM GDP in the presence of 1 mM Ca²⁺. Valueswere normalized to 1 mM Ca²⁺ in the absence of other metals. Emissionmaxima at 510 nm; excitation at 488 nm.

FIG. 21D shows the stopped-flow fluorescence using 10 μM CatchER atvarious Ca²⁺ concentrations recorded at 395 nm excitation. CatchER'sfluorescence response in 0 mM Ca²⁺ was measured as the baseline.

FIG. 21E shows the stopped-flow traces showing decreased fluorescenceupon rapid mixture of Ca²⁺-loaded CatchER with 200 μM EGTA. Emission at510 nm.

FIG. 22A shows a representative chemical shift of cross-peak Y143 at[Ca²⁺]=0, 0.5, 1, 2, 4, and 6 mM, overlaid with 2D [¹H-¹⁵N] HSQC spectraof 0.3 mM CatchER in response to Ca²⁺.

FIG. 22B shows a Q69 chemical shift perturbation induced by Ca²⁺titration. A minor peak was separated from the original single peakafter adding 2 mM Ca²⁺, and the ratio of integration of peak b to peak aincreased from 0 to 2.27 as Ca²⁺ concentration increased from 1 mM to 6mM.

FIG. 22C shows combined chemical shift changes in combining a backboneamide proton and nitrogen between the Ca²⁺-saturated and Ca²⁺-free form.Ca²⁺ influences the residues interacting with the chromophore or closeto the designed Ca²⁺ binding site. Y182, highly accessible to solvents,and G228 in the flexible C-terminal also exhibited more than a 0.2-ppmchange in chemical shift. The secondary structure of CatchER, accordingto EGFP, was labeled on the top. All data were recorded at 37° C. usinga 600 MHz NMR spectrometer with 300 μM ¹⁵N-labeled samples in 10 mMTris, 10 mM KCl, pH 7.4.

FIG. 23A illustrates C2C12 myoblast endoplasmic reticulum Ca²⁺ dynamicsmonitored with CatchER. Two representative fluorescence responses tointact myoblasts without extracellular Ca²⁺ or EGTA were evoked by 100μM ATP (pH 7.0) twice separated by a Ringer buffer washout.

FIG. 23B illustrates the same batch of cells as in FIG. 23A whenpermeabilized with 25 μM digitonin in intracellular buffer for 3 minsand sequentially treated with IP₃, intracellular buffer washout,thapsigargin, IP₃ (arrow), washout (triangle), and ionomycin (arrow).

FIG. 23C illustrates representative fluorescent imaging of C2C12co-expressing CatchER and mCherry-ER.

FIG. 23D illustrates CatchER (top) and mCherry-ER (bottom) fluorescenceresponses to 4-Chloro-m-Cresol (4-CmC) application. Time points ofcorresponding imaging in FIG. 23C are indicated.

FIG. 23E illustrates 4-CmC evoked Ca²⁺ release in the absence andpresence of thapsigargin.

FIG. 24 illustrates 4-CmC evoked cytosolic Ca²⁺ changes detected byFura-2.

FIGS. 25A-25N illustrate the fluorescence and UV-absorbance changes ofpurified bacterially expressed EGFP-based sensors in response to Ca²⁺demonstrating adjustments of the sensor dynamic ranges.

FIG. 25A illustrates overlaid absorbance spectra from 220 nm to 600 nmof EGFP in the presence of 10 μM EGTA (solid line) or 5 mM Ca²⁺ (dashedline).

FIGS. 25B-25F illustrate absorbance spectra from 220 nm to 600 nm ofEGFP-based sensors D8, D9, D10, CatchER, and D12 in the presence of 10μM EGTA (solid lines) or 5 mM Ca²⁺ (dashed lines). The absorbance maximaat 488 nm increased and 395 nm decreased for D9, D10, and CatchER (C-E)in response to Ca²⁺.

FIG. 25G illustrates overlaid fluorescence emission spectra from 500 nmto 600 nm of EGFP measured by a fluorometer in the presence of 10 μMEGTA (solid line) or 5 mM Ca²⁺ (dashed line). The two overlaid emissionspectra on the top were excited at 488 nm and the two or at 395 nm(bottom).

FIGS. 25H-25L illustrate the fluorescent emission spectra of theEGFP-based sensors D8, D9, D10, CatchER and D12, respectively.

FIG. 25M is a graph showing the comparison of the amplitudes offluorescence emission change at 510 nm excited at 488 nm (black bar) and395 nm (gray bar) of EGFP and designed variants in response to Ca²⁺. Theamplitude change is in the term of (F_(Holo)/F_(Apo)-1), and F_(Holo)and F_(Apo) represent the absorbance intensity in the presence of 5 mMCa²⁺ and 10 μM EGTA, respectively. The non-ratiometric fluorescencechange at 510 nm excited at either 488 nm and 395 nm of D9, D10, CatchERand D12 is presented in the positive values of the bars.

FIG. 25N is a graph showing the comparison of the amplitudes ofabsorbance change at 488 nm (black bar) and 395 nm (gray bar) of EGFPand variants thereof in response to Ca²⁺. The amplitude change is in theterm of (A_(Holo)/A_(Apo)-1), and A_(Holo) and A_(Apo) represent theabsorbance intensity in the presence of 5 mM Ca²⁺ and 10 μM EGTA,respectively. The ratiometric absorbance change at 488 nm and 395 nm ofD9, D10 and CatchER in response to Ca²⁺ is presented in the positive andnegative values of the bars, respectively. Absorbance at 280 nm of allthe variants was normalized to 1.

FIGS. 26A-26G illustrate the pH stability of CatchER before and afterbinding Ca²⁺ as determined by measuring the apparent pKa values based onpH-dependence of the fluorescence intensity, and the stoichiometricinteraction between CatchER and Ca²⁺ is determined by Job's Plot.

FIG. 26A shows the fluorescence emission intensities at 510 nm wererecorded in the presence of 10 μM EGTA (circle) or 4 mM Ca²⁺ (square)with excitation at 488 nm at corresponding pH values. The apparent pKawas 7.59±0.03 (EGTA) and 6.91±0.03 (Ca²⁺).

FIG. 26B shows the pH-dependence of the fluorescence emissionintensities at 510 nm excited at 395 nm. The apparent pKa was 7.14±0.02(EGTA) and 6.95±0.06 (Ca²⁺).

FIG. 26C shows a Job's Plot of the relative amount of Ca²⁺-bound CatchERas determined by fluorescence (F₄₈₈, F₃₉₅) and absorbance (A₄₈₈) as afunction of the concentration of CatchER.

FIG. 26D shows the numerical results of the Job Plot of FIG. 26C.

FIG. 26E shows fluorescent spectra with the concentration of CatchER inμM 28.7, 23.3, 19.4, 15.1, and 11.6 (solid line) in response to [Ca²⁺](in μM)=11.3, 16.7, 20.6, 24.9, 28.4 (dashed line), excited at 488 nm.

FIG. 26F shows fluorescent spectra with the concentration of CatchER inμM 28.7, 23.3, 19.4, 15.1, and 11.6 (solid line) in response to [Ca²⁺](in μM)=11.3, 16.7, 20.6, 24.9, 28.4 (dashed line), excited at 395 nm.

FIG. 26G shows the corresponding absorbance change in the absence (solidline) or presence (dashed line) of Ca²⁺.

FIGS. 27A and 27B illustrate Ca²⁺ binding by CatchER by equilibriumdialysis and an Inductively Coupled Plasma Optical Emission Spectrometer(ICP-OES).

FIG. 27A shows representative spectra of ICP-OES (Inductively CoupledPlasma Optical Emission Spectrometry) to determine the total Ca²⁺concentration (bound and unbound) outside a dialysis tube (buffer) andinside the dialysis tube with the samples of myoglobin, EGFP, CatchERand α-lactalbumin, respectively, with maximal intensity at 370.602 nm.Each spectrum is the average of three-time repeats with the error bars,and the amplitude of peak intensity of each sample represents theconcentration of Ca²⁺.

FIG. 27B shows the comparison of Ca²⁺ concentration of each sampledetermined by ICP-OES. The peak intensities recorded at 396.847,373.690, 219.779, 370.602, 317.933, 643.907 and 220.861 nm wereconverted to Ca²⁺ concentration calibrated by the pre-determined Ca²⁺standard linear curve at each wavelength, respectively. The Ca²⁺concentration of the buffer outside the dialysis tube was 60.4±0.7 μM(unbound), and inside (both bound and unbound), containing myoglobin,EGFP, CatchER and α-lactalbumin was 61.5±1.2, 64.5±1.1, 74.6±1.5 and79.1±1.7 μM (both bound and unbound), respectively.

FIGS. 28A-28C illustrate the monomerization of CatchER by measuredrotational correlation time τ_(C) with high-field nuclear magneticresonance spectroscopy.

FIG. 28A shows τ_(C) directly determined by the SCT-CCR experimentperformed on an 800 MHz NMR spectrometer (gray square) or calculatedusing Eq. (16) and (17) with relaxation times T₁, T₂ determined on a 600MHz NMR spectrometer (black circle). The secondary structures ofcorresponding residues are marked above.

FIG. 28B shows representative fitting of peaks integrations collected at0, 30, 60, 100, 240, 480, 720, 1000, and 1500 ms T₁ delays.

FIG. 28C shows overlay of T1 delay spectra from selected region: 0 msand 1500 ms.

FIGS. 29A-29H illustrate CatchER NMR assignment and Ca²⁺ influence onresidues interacting with the chromophore on the opposite side of thedesigned Ca²⁺ binding site.

FIG. 29A shows selected CatchER 3D HNCA spectra from I14 to E17, withsequential and intraresidual Cα-Cα connections indicated by red lines.

FIG. 29B shows a CatchER 2D {¹H-¹⁵N} HSQC spectrum.

FIG. 29C shows Cα chemical shifts. Most labeled residues exhibiting morethan a 1.5 p.p.m. chemical shift difference were sequentially close tothe chromophore or the designed Ca²⁺ binding site. Nos. 1-5 representE147, D202, E204, E223, and E225, respectively. All the data wererecorded at 37° C. using an 800 MHz NMR spectrometer with a cryogenicprobe and a 300 mM ¹³C-¹⁵N double-labeled sample in 10 mM Tris, pH 7.4.

FIGS. 29D-29G show CatchER 2D {¹H-¹⁵N} HSQC spectrum recorded at 0 mMCa²⁺ (black) and 6 mM Ca²⁺ (red). A chemical shift change was observedfor Q94 at 6 mM Ca²⁺ but no change for R96, F165, or V61.

FIG. 29H shows sidechains of R96, Q94, F165, and V61 protruding towardthe chromophore on the opposite side of the designed Ca²⁺ binding site.Data were recorded at 37° C. using a 600 MHz NMR spectrometer with a 300μM ¹⁵N labeled sample in 10 mM Tris and 10 mM KCl, pH 7.4.

FIGS. 30A-30B illustrate the localization of CatchER expressed in the ERof HEK-293 and C2C12 cells and SR of FDB fibers.

FIG. 30A shows colocalization of CatchER and DsRed2-ER in HEK-293 cells.CatchER and DsRed2-ER (were transiently co-transfected and expressed intwo cell lines for confocal microscopy imaging. The overlay imagingshows the colocalization of CatchER corresponding to ER-trackerDsRed2-ER.

FIG. 30B shows co-localization of CatchER and DsRed2-ER in C2C12 cells.CatchER and DsRed2-ER (were transiently co-transfected and expressed intwo cell lines for confocal microscopy imaging. The overlay imagingshows the co-localization of CatchER corresponding to ER-trackerDsRed2-ER.

FIGS. 31A-31I illustrate the in situ determination of K_(d) andendoplasmic reticulum Ca²⁺ dynamics of HeLa and HEK293 cells.

FIG. 31A shows in situ determination of K_(d) in ER of C2C12 myoblastcells. 1-5 correspond to 1, 3, 10, and 20 mM Ca²⁺ and 3 mM EGTA,respectively.

FIG. 31B shows a K_(d) determination in BHK cells. 1-7 represent 0.05,0.1, 0.5, 1, 5, and 10 mM Ca²⁺ and 200 μM EGTA. CatchER fluorescentsignals of transfected permeabilized cells after equilibration withvarious extracellular Ca²⁺ concentrations excited at 488 nm.

FIG. 31C shows a K_(d) calculation with a 1:1 binding mode.

FIG. 31D shows a representative ER Ca²⁺ signaling detected by CatchER inHeLa cells triggered by ATP.

FIG. 31E shows a representative ER Ca²⁺ signaling detected by CatchER inHeLa cells triggered by histamine.

FIG. 31F shows a representative ER Ca²⁺ signaling detected by CatchER inHeLa cells triggered by CPA.

FIG. 31G shows a representative ER Ca²⁺ signaling detected by CatchER inHeLa cells triggered by ATP.

FIG. 31H reversible Ca²⁺ release triggered by 50 μM histamine in HEK293cells.

FIG. 31I shows quantification of irreversible ER Ca²⁺ release in HEK293cells induced by 2 μM thapsigargin in the presence of 1 mM extracellularCa²⁺. F_(min) and F_(max) were determined by adding 5 mM EGTA and 50 mMCa²⁺, respectively, to the intact cells in the presence of 5 μMionomycin (n=6).

FIG. 32 shows the temperature dependent NMR HSQC spectra changes ofCatchER.

FIG. 33 shows a 1D NMR spectra of chemical shift changes of CatchERtriggered by Ca²⁺.

FIG. 34 illustrates X-ray crystal structures of chromophore conformationchange of Apo_CatchER and Ca²⁺_loaded CatchER, and correlated absorptionspectra. (red is light grey, blue is dark grey, green is medium grey,cyan is light grey)

FIG. 35 illustrates X-ray crystal structures of chromophore conformationchange of Apo_CatchER, Ca²⁺_loaded CatchER, and Gd³⁺_loaded CatchER.(cyan is light grey, purple is dark grey, green is medium grey)

The drawings are described in greater detail in the description andexamples below.

The details of some exemplary embodiments of the methods and systems ofthe present disclosure are set forth in the description below. Otherfeatures, objects, and advantages of the disclosure will be apparent toone of skill in the art upon examination of the following description,drawings, examples and claims. It is intended that all such additionalsystems, methods, features, and advantages be included within thisdescription, be within the scope of the present disclosure, and beprotected by the accompanying claims.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise. In this disclosure, “comprises,”“comprising,” “containing” and “having” and the like can have themeaning ascribed to them in U.S. patent law and can mean “includes,”“including,” and the like; “consisting essentially of” or “consistsessentially” or the like, when applied to methods and compositionsencompassed by the present disclosure refers to compositions like thosedisclosed herein, but which may contain additional structural groups,composition components or method steps (or analogs or derivativesthereof as discussed above). Such additional structural groups,composition components or method steps, etc., however, do not materiallyaffect the basic and novel characteristic(s) of the compositions ormethods, compared to those of the corresponding compositions or methodsdisclosed herein. “Consisting essentially of” or “consists essentially”or the like, when applied to methods and compositions encompassed by thepresent disclosure have the meaning ascribed in U.S. patent law and theterm is open-ended, allowing for the presence of more than that which isrecited so long as basic or novel characteristics of that which isrecited is not changed by the presence of more than that which isrecited, but excludes prior art embodiments.

DEFINITIONS

In describing and claiming the disclosed subject matter, the followingterminology will be used in accordance with the definitions set forthbelow.

Further definitions are provided in context below. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art ofmolecular biology. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described herein.

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which thisdisclosure pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. The techniques and procedures described orreferenced herein are generally well understood and commonly employedusing conventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition(2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. andCurrent Protocols in Molecular Biology (Ausbel et al., eds., John Wiley& Sons, Inc. 2001). As appropriate, procedures involving the use ofcommercially available kits and reagents are generally carried out inaccordance with manufacturer defined protocols and/or parameters unlessotherwise noted.

The term “polypeptide” as used herein refers to proteins and fragmentsthereof. Polypeptides are disclosed herein as amino acid residuesequences. Those sequences are written left to right in the directionfrom the amino to the carboxy terminus. In accordance with standardnomenclature, amino acid residue sequences are denominated by either athree letter or a single letter code as indicated as follows: Alanine(Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp,D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E),Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu,L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F),Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp,W), Tyrosine (Tyr, Y), and Valine (Val, V).

The term “variant” as used herein refers to a polypeptide orpolynucleotide that differs from a reference polypeptide orpolynucleotide, but retains essential properties. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overall(homologous) and, in many regions, identical. A variant and referencepolypeptide may differ in amino acid sequence by one or moremodifications (e.g., substitutions, additions, and/or deletions). Asubstituted or inserted amino acid residue may or may not be one encodedby the genetic code. A variant of a polypeptide may be naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally.

Modifications and changes can be made in the structure of thepolypeptides of this disclosure and still result in a molecule havingsimilar characteristics as the polypeptide (e.g., a conservative aminoacid substitution). For example, certain amino acids can be substitutedfor other amino acids in a sequence without appreciable loss ofactivity. Because it is the interactive capacity and nature of apolypeptide that defines that polypeptide's biological functionalactivity, certain amino acid sequence substitutions can be made in apolypeptide sequence and nevertheless obtain a polypeptide with likeproperties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionof amino acids whose hydropathic indices are within ±2 is preferred,those within ±1 are particularly preferred, and those within ±0.5 areeven more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biologically functionalequivalent polypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take one or more of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include, but are not limited to (original residue: exemplarysubstitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu,Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile:Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr),(Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu).Embodiments of this disclosure thus contemplate functional or biologicalequivalents of a polypeptide as set forth above. In particular,embodiments of the polypeptides can include variants having about 50%,60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide ofinterest.

The term “identity,” as used herein refers to a relationship between twoor more polypeptide sequences, as determined by comparing the sequences.In the art, “identity” also refers to the degree of sequence relatednessbetween polypeptide as determined by the match between strings of suchsequences. “Identity” and “similarity” can be readily calculated byknown methods, including, but not limited to, those described inComputational Molecular Biology, Lesk, A. M., Ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073, (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs. Thepercent identity between two sequences can be determined by usinganalysis software (i.e., Sequence Analysis Software Package of theGenetics Computer Group, Madison Wis.) that incorporates the Needelmanand Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST,and XBLAST). The default parameters are used to determine the identityfor the polypeptides of the present invention.

By way of example, a polypeptide sequence may be identical to thereference sequence, that is be 100% identical, or it may include up to acertain integer number of amino acid alterations as compared to thereference sequence such that the % identity is less than 100%. Suchalterations are selected from: at least one amino acid deletion,substitution (including conservative and non-conservative substitution),or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminus positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence, or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in the reference polypeptideby the numerical percent of the respective percent identity (divided by100) and then subtracting that product from said total number of aminoacids in the reference polypeptide.

Conservative amino acid variants can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine,allo-threonine, methylthreonine, hydroxy-ethylcysteine,hydroxyethylhomocysteine, nitro-glutamine, homoglutamine, pipecolicacid, thiazolidine carboxylic acid, dehydroproline, 3- and4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline,2-azaphenyl-alanine, 3-azaphenylalanine, 4-azaphenylalanine, and4-fluorophenylalanine. Several methods are known in the art forincorporating non-naturally occurring amino acid residues into proteins.For example, an in vitro system can be employed wherein nonsensemutations are suppressed using chemically aminoacylated suppressortRNAs. Methods for synthesizing amino acids and aminoacylating tRNA areknown in the art. Transcription and translation of plasmids containingnonsense mutations is carried out in a cell-free system comprising an E.coli S30 extract and commercially-available enzymes and other reagents.Proteins are purified by chromatography. (Robertson et al., (1991) J.Am. Chem. Soc. 113: 2722; Ellman et al., (1991) Methods Enzymol. 202:301; Chung et al., Science (1993) 259: 806-809; and Chung et al., (1993)Proc. Natl. Acad. Sci. USA, 90: 10145-10149). In a second method,translation is carried out in Xenopus oocytes by microinjection ofmutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti etal., (1996) J. Biol. Chem. 271: 19991-19998). Within a third method, E.coli cells are cultured in the absence of a natural amino acid that isto be replaced (e.g., phenylalanine) and in the presence of the desirednon-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Thenon-naturally occurring amino acid is incorporated into the protein inplace of its natural counterpart. (Koide et al., (1994) Biochem. 33:7470-7476). Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn et al., (1993) ProteinSci. 2: 395-403).

The term “polynucleotide” as used herein refers to anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotidesas used herein refers to, among others, single- and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. The terms “nucleic acid,”“nucleic acid sequence,” or “oligonucleotide” also encompass apolynucleotide as defined above.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically, or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

By way of example, a polynucleotide sequence of the present disclosuremay be identical to the reference sequence, that is be 100% identical,or it may include up to a certain integer number of nucleotidealterations as compared to the reference sequence. Such alterations areselected from the group including at least one nucleotide deletion,substitution, including transition and transversion, or insertion, andwherein said alterations may occur at the 5′ or 3′ terminus positions ofthe reference nucleotide sequence or anywhere between those terminuspositions, interspersed either individually among the nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The number of nucleotide alterations is determinedby multiplying the total number of nucleotides in the referencenucleotide by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number ofnucleotides in the reference nucleotide. Alterations of a polynucleotidesequence encoding the polypeptide may alter the polypeptide encoded bythe polynucleotide following such alterations.

As used herein, DNA may obtained by any method. For example, the DNAincludes complementary DNA (cDNA) prepared from mRNA, DNA prepared fromgenomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCRamplification with RNA or DNA as a template, and DNA constructed byappropriately combining these methods.

cDNA can be cloned from mRNA encoding the protein by, for example, themethod described below:

First, the mRNA encoding the protein is prepared from theabove-mentioned tissues or cells expressing and producing the protein.mRNA can be prepared by isolating total RNA by a known method such asguanidine-thiocyanate method (Chirgwin et al., Biochemistry, 18:5294,1979), hot phenol method, or AGPC method, and subjecting it to affinitychromatography using oligo-dT cellulose or poly-U Sepharose.

Then, with the mRNA obtained as a template, cDNA is synthesized, forexample, by a well-known method using reverse transcriptase, such as themethod of Okayama et al (Mol. Cell. Biol. 2:161 (1982); Mol. Cell. Biol.3:280 (1983)) or the method of Hoffman et al. (Gene 25:263 (1983)), andconverted into double-stranded cDNA. A cDNA library is prepared bytransforming E. coli with plasmid vectors, phage vectors, or cosmidvectors having this cDNA or by transfecting E. coli after in vitropackaging.

As used herein, an “isolated nucleic acid” is a nucleic acid, thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. For example, the substantially purepolypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight.Purity can be measured by any appropriate standard method known in theart, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

The plasmid vectors used herein are not limited as long as they arereplicated and maintained in hosts. Any phage vector that can bereplicated in hosts can also be used. Examples of commonly used cloningvectors are pUC19, λgt10, λgt11, and so on. When the vector is appliedto immunological screening as mentioned below, a vector having apromoter that can express a gene encoding the desired protein in a hostis preferably used.

cDNA can be inserted into a plasmid by, for example, the method ofManiatis et al. (Molecular Cloning, A Laboratory Manual, second edition,Cold Spring Harbor Laboratory, p. 1.53, 1989). cDNA can be inserted intoa phage vector by, for example, the method of Hyunh et al. (DNA cloning,a practical approach, 1, p. 49 (1985)). These methods can be simplyperformed by using a commercially available cloning kit (for example, aproduct from Takara Shuzo). The recombinant plasmid or phage vector thusobtained is introduced into an appropriate host cell such as aprokaryote (for example, E. coli strains HB101, DH5a, MC1061/P3, etc).

Examples of a method for introducing a plasmid into a host are, calciumchloride method, calcium chloride/rubidium chloride method, lipidsomemethod, and electroporation method, described in Molecular Cloning, ALaboratory Manual (second edition, Cold Spring Harbor Laboratory, p.1.74 (1989)). Phage vectors can be introduced into host cells by, forexample, a method in which the phage DNAs are introduced into grownhosts after in vitro packaging. In vitro packaging can be easilyperformed with a commercially available in vitro packaging kit (forexample, a product from Stratagene or Amersham).

The identification of cDNA encoding protein, its expression beingaugmented depending on the stimulation of cytokines like AID proteindisclosed herein, can be carried out by for example suppression subtracthybridization (SSH) ((1996) Proc. Natl. Acad. Sci. USA. 93: 6025-6030;Anal. Biochem. (1996) 240: 90-97) taking advantage of suppressive PCReffect ((1995) Nucleic Acids Res. 23:1087-1088) using two cDNAlibraries, namely, cDNA library constructed from mRNA derived fromstimulated cells (tester cDNA library) and that constructed from mRNAderived from unstimulated cells (driver cDNA library).

Embodiments of the present disclosure relate to a recombinant vectorcomprising the DNA encoding the protein used herein. As a recombinantvector disclosed herein, any vector can be used as long as it is capableof retaining replication or self-multiplication in each host cell ofprokaryotic and/or eukaryotic cells, including plasmid vectors and phagevectors. The recombinant vector can easily be prepared by ligating theDNA encoding protein with a vector for recombination available in theart (plasmid DNA and bacteriophage DNA) by the usual method.

Specific examples of the vectors for recombination used are E.coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19,yeast-derived plasmids such as pSH19 and pSH15, and Bacillussubtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples ofphages are a bacteriophage such as lambda phage, and an animal or insectvirus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, andnuclear polyhedrosis virus.

An “expression vector” is useful for expressing the DNA encoding theprotein used herein and for producing the protein. The expression vectoris not limited as long as it expresses the gene encoding the protein invarious prokaryotic and/or eukaryotic host cells and produces thisprotein. Examples thereof are pMAL C2, pEF-BOS ((1990) Nucleic AcidsRes. 18:5322, and so on), pME18S pCDNA (Experimental Medicine:SUPPLEMENT, “Handbook of Genetic Engineering” (1992)), etc.

When bacteria, particularly E. coli, are used as host cells anexpression vector generally comprises, at least, a promoter/operatorregion, an initiation codon, the DNA encoding the protein terminationcodon, terminator region, and replicon.

When yeast, animal cells, or insect cells are used as hosts, anexpression vector is preferably comprised of, at least; a promoter, aninitiation codon, the DNA encoding the protein and a termination codon.It may also comprise the DNA encoding a signal peptide, enhancersequence, 5′- and 3′-untranslated region of the gene encoding theprotein, splicing junctions, polyadenylation site, selectable markerregion, and replicon. The expression vector may also contain, ifrequired, a gene for gene amplification (marker) that is usually used.DNA plasmids can also be directly introduced to the mammalian cells ofanimals to express proteins.

A promoter/operator region to express the protein in bacteria comprisesa promoter, an operator, and a Shine-Dalgarno (SD) sequence (forexample, AAGG). For example, when the host is Escherichia, it preferablycomprises Trp promoter, lac promoter, recA promoter, APL promoter, tacpromoter, or the like. Examples of a promoter to express the protein inyeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, andso on. When the host is Bacillus, examples thereof are SL01 promoter,SP02 promoter, penP promoter, and so on. When the host is a eukaryoticcell such as a mammalian cell, examples thereof are SV40-derivedpromoter, retrovirus promoter, heat shock promoter, and so on, andpreferably SV-40 and retrovirus-derived one. As a matter of course, thepromoter is not limited to the above examples. In addition, using anenhancer is effective for expression.

A preferable initiation codon is, for example, a methionine codon (ATG).

A commonly used termination codon (for example, TAG, TAA, TGA) isexemplified as a termination codon. Usually, used natural or syntheticterminators are used as a terminator region.

A “replicon” means a DNA capable of replicating the whole DNA sequencein host cells, and includes a natural plasmid, an artificially modifiedplasmid (DNA fragment prepared from a natural plasmid), a syntheticplasmid, and so on. Examples of preferable plasmids are pBR322 or itsartificial derivatives (DNA fragment obtained by treating pBR322 orpRSET with appropriate restriction enzymes) for E. coli, yeast 2μplasmid or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198,pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, andsuch for mammalian cells.

An enhancer sequence, polyadenylation site, and splicing junction thatare usually used in the art, such as those derived from SV40 can also beused.

A selectable marker usually employed can be used according to the usualmethod. Examples thereof are resistance genes for antibiotics, such astetracycline, ampicillin, or kanamycin.

Examples of genes for gene amplification are dihydrofolate reductase(DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamatesynthase gene, adenosine deaminase gene, ornithine decarboxylase gene,hygromycin-B-phosphotransferase gene, aspartate transcarbamylase gene,etc.

The expression vector used herein can be prepared by continuously andcircularly linking at least the above-mentioned promoter, initiationcodon, DNA encoding the protein, termination codon, and terminatorregion, to an appropriate replicon. If desired, appropriate DNAfragments (for example, linkers, restriction sites, and so on), can beused by the usual method such as digestion with a restriction enzyme orligation using T4 DNA ligase.

Affinity tags such His-tag and GST can be added at the sequence end tofacilitate protein purification and recognition by Western blot andpulldown assay. Examples of other tags such as HA and FLAG can also beadded to allow further manipulation of the constructs.

As used herein, “transformants” can be prepared by introducing theexpression vector mentioned above into host cells.

As used herein, “host” cells are not limited as long as they arecompatible with an expression vector mentioned above and can betransformed. Examples thereof are various cells such as wild-type cellsor artificially established recombinant cells usually used in technicalfield (for example, bacteria (Escherichia and Bacillus), yeast(Saccharomyces, Pichia, and such), animal cells, or insect cells).

E. coli or animal cells are preferably used. Specific examples are E.coli strains DH5 alpha, TB1, HB101, and the like, mouse-derived cells(COP, L, C127, Sp2/0, NS-1, NIH 3T3, and such), rat-derived cells (PC12,PC12h), hamster-derived cells (BHK, CHO, and such), monkey-derived cells(COS1, COS3, COS7, CV1, Velo, and such), and human-derived cells (Hela,diploid fibroblast-derived cells, myeloma cells, and HepG2, and such).

An expression vector can be introduced (transformed (transfected)) intohost cells by known methods. Transformation can be performed, forexample, according to the method of Cohen et al. ((1972) Proc. Natl.Acad. Sci. USA. 69: 2110), protoplast method ((1979) Mol. Gen. Genet.168: 111), or competent method ((1971) J. Mol. Biol. 56: 209) when thehosts are bacteria (E. coli, Bacillus subtilis, and the like), themethod of Hinnen et al. ((1978) Proc. Natl. Acad. Sci. USA. 75: 1927),or lithium method ((1983) J. Bacteriol. 153: 163) when the host isSaccharomyces cerevisiae, the method of Graham ((1973) Virology 52: 456)when the hosts are animal cells, and the method of Summers et al.((1983) Mol. Cell. Biol. 3: 2156-2165) when the hosts are insect cells.

The protein disclosed herein, can be produced by cultivatingtransformants (in the following, this term includes transfectants)comprising an expression vector prepared as mentioned above in nutrientmedia.

The nutrient media preferably comprise a carbon source, an inorganic ororganic nitrogen source necessary for the growth of host cells(transformants). Examples of the carbon source are glucose, dextran,soluble starch, and sucrose, and examples of the inorganic or organicnitrogen source are ammonium salts, nitrates, amino acids, corn steepliquor, peptone, casein, meat extract, soy bean cake, and potatoextract. If desired, they may comprise other nutrients (for example, aninorganic salt (for example, calcium chloride, sodiumdihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (forexample, tetracycline, neomycin, ampicillin, kanamycin, and so on).

Cultivation of cell lines is performed by a method known in the art.Cultivation conditions such as temperature, pH of the media, andcultivation time are selected appropriately so that the protein isproduced in large quantities.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically, or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

By way of example, a polynucleotide sequence of the present disclosuremay be identical to the reference sequence, that is be 100% identical,or it may include up to a certain integer number of nucleotidealterations as compared to the reference sequence. Such alterations areselected from the group including at least one nucleotide deletion,substitution, including transition and transversion, or insertion, andwherein said alterations may occur at the 5′ or 3′ terminus positions ofthe reference nucleotide sequence or anywhere between those terminuspositions, interspersed either individually among the nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The number of nucleotide alterations is determinedby multiplying the total number of nucleotides in the referencenucleotide by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number ofnucleotides in the reference nucleotide. Alterations of a polynucleotidesequence encoding the polypeptide may alter the polypeptide encoded bythe polynucleotide following such alterations.

The term “codon” means a specific triplet of mononucleotides in the DNAchain or mRNA that make up an amino acid or termination signal.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (e.g., GAU and GAC triplets each encodeAsp).

As used herein, the term “exogenous DNA” or “exogenous nucleic acidsequence” or “exogenous polynucleotide” refers to a nucleic acidsequence that was introduced into a cell or organelle from an externalsource. Typically the introduced exogenous sequence is a recombinantsequence.

As used herein, the term “transfection” refers to the introduction of anucleic acid sequence into the interior of a membrane enclosed space ofa living cell, including introduction of the nucleic acid sequence intothe cytosol of a cell as well as the interior space of a mitochondria,nucleus or chloroplast. The nucleic acid may be in the form of naked DNAor RNA, associated with various proteins, or the nucleic acid may beincorporated into a vector.

“DNA regulatory sequences”, as used herein, are transcriptional andtranslational control sequences, such as promoters, enhancers,polyadenylation signals, termination signals, and the like, that providefor and/or regulate expression of a coding sequence in a host cell.

A “promoter sequence” is a DNA regulatory region in an operon capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. The promoter sequence isbound at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at levels detectableabove background. Within the promoter sequence will be found atranscription initiation site, as well as protein binding domainsresponsible for the binding of RNA polymerase. Eukaryotic promoters willoften, but not always, contain “TATA” boxes and “CAT” boxes. Variouspromoters, including inducible promoters, may be used to drive thevarious vectors of the present disclosure.

The terms “chimeric”, “fusion” and “composite” are used to denote aprotein, peptide domain or nucleotide sequence or molecule containing atleast two component portions that are mutually heterologous in the sensethat they are not, otherwise, found directly (covalently) linked innature. More specifically, the component portions are not found in thesame continuous polypeptide or gene in nature, at least not in the sameorder or orientation or with the same spacing present in the chimericprotein or composite domain. Such materials contain components derivedfrom at least two different proteins or genes or from at least twonon-adjacent portions of the same protein or gene. Composite proteins,and DNA sequences that encode them, are recombinant in the sense thatthey contain at least two constituent portions that are not otherwisefound directly linked (covalently) together in nature.

The term “domain” in this context is not intended to be limited to asingle discrete folding domain.

A “reporter polynucleotide” includes any gene that expresses adetectable gene product, which may be RNA or a reporter polypeptide.Reporter genes include coding sequences for which the transcriptionaland/or translational products are readily detectable or selectable.

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition or insertionof one or more amino acid or nucleotide residues, respectively, ascompared to the corresponding naturally occurring molecule.

A “deletion” or “subtraction”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the deletion orsubtraction of one or more amino acid or nucleotide residues,respectively, as compared to the corresponding naturally occurringmolecule.

A “substitution”, as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

A “mutation” is an inheritable change in a DNA sequence relative to areference “wild-type” DNA sequence. Mutations can occur as a result of asingle base change, multiple base changes, or the addition or deletionof more than one nucleotide to a DNA sequence.

The term “mutant” is employed broadly to refer to a protein that differsin some way from a reference wild-type protein, where the protein mayretain biological properties of the reference wild-type (e.g., naturallyoccurring) protein, or may have biological properties that differ fromthe reference wild-type protein. The term “biological property” of thesubject proteins includes, but is not limited to, spectral properties,such as emission maximum, quantum yield, and brightness, and the like;in vivo and/or in vitro stability (e.g., half-life); and the like.Mutants can include single amino acid changes (point mutations),deletions of one or more amino acids (point-deletions), N-terminaltruncations, C-terminal truncations, insertions, and the like. Mutantscan be generated using standard techniques of molecular biology.

A “gene mutation” refers to a mutation that occurs entirely within onegene, or its upstream regulatory sequences and can comprise either apoint mutation or other disruption of normal chromosomal structure thatoccurs entirely within one gene.

A “wild-type” strain is capable of a full range of metabolic activities.For example, wild-type strains of Salmonella can synthesize all 20 aminoacids from a single carbon source.

A “mutant” strain is not capable of all of the activities of thewild-type strain from which it is derived. For example, a mutantbacterial strain that is defective in its ability to synthesize theamino acid histidine (his strain) requires the presence of exogenoushistidine in order to grow.

A “point mutation” is a change in one, or a small number of base pairs,in a DNA sequence. Point mutations may result from base pairsubstitutions or from small insertions or deletions.

A “transition” is a point mutation in which a purine is replaced with apurine or a pyrimidine is replaced with a pyrimidine.

A “transversion” is a point mutation in which a purine is replaced witha pyrimidine or a pyrimidine with a purine. Generally speaking,transitions are more common than transversions because the former arenot detected by the proofreading enzymes.

Alternatively, point mutation can also cause a nonsense mutationresulting from the insertion of a stop codon (amber, ochre, opal). Basepair mutations that generate a translation stop codon causes prematuretermination of translation of the coded protein.

A “frameshift mutation” results from the insertion or deletion of one ormore nucleotides within a gene. The “reading frame” of a gene refers tothe order of the bases with respect to the starting point fortranslation of the mRNA. Deletion of a single base pair results inmoving ahead one base in all of the codons, and is often referred to asa positive frameshift. Addition of one base pair (or loss of two basepairs) shifts the reading frame behind by one base, and is oftenreferred to as a negative frameshift.

As used herein, the term “hybridization” refers to the process ofassociation of two nucleic acid strands to form an antiparallel duplexstabilized by means of hydrogen bonding between residues of the oppositenucleic acid strands.

“Hybridizing” and “binding”, with respect to polynucleotides, are usedinterchangeably. The terms “hybridizing specifically to” and “specifichybridization” and “selectively hybridize to,” as used herein refer tothe binding, duplexing, or hybridizing of a nucleic acid moleculepreferentially to a particular nucleotide sequence under stringentconditions.

The term “stringent assay conditions” as used herein refers toconditions that are compatible to produce binding pairs of nucleicacids, e.g., surface bound and solution phase nucleic acids, ofsufficient complementarity to provide for the desired level ofspecificity in the assay while being less compatible to the formation ofbinding pairs between binding members of insufficient corn plementarityto provide for the desired specificity. Stringent assay conditions arethe summation or combination (totality) of both hybridization and washconditions.

In accordance with the present disclosure, “a detectably effectiveamount” of the sensor of the present disclosure is defined as an amountsufficient to yield an acceptable image using equipment that isavailable for clinical use. A detectably effective amount of the sensorof the present disclosure may be administered in more than oneinjection. The detectably effective amount of the sensor of the presentdisclosure can vary according to factors such as the degree ofsusceptibility of the individual, the age, sex, and weight of theindividual, idiosyncratic responses of the individual, the dosimetry,and the like. Detectably effective amounts of the sensor of the presentdisclosure can also vary according to instrument and film-relatedfactors. Optimization of such factors is well within the level of skillin the art.

By “administration” is meant introducing a sensor of the presentdisclosure into a subject. The preferred route of administration of thesensor is intravenous. However, any route of administration, such asoral, topical, subcutaneous, peritoneal, intraarterial, inhalation,vaginal, rectal, nasal, introduction into the cerebrospinal fluid, orinstillation into body compartments can be used.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, an antibody, or a host cell that is in anenvironment different from that in which the polynucleotide, thepolypeptide, the antibody, or the host cell naturally occurs.

As used herein the phrase “beta-can structure of proteins” refers to aprotein featured as a compact cylinder is formed with antiparallel betastrands.

“Fluorescent protein” refers to any protein capable of emitting lightwhen excited with appropriate electromagnetic radiation. Fluorescentproteins include proteins having amino acid sequences that are eithernatural or engineered, such as the fluorescent proteins derived fromAequorea-related fluorescent proteins. A “fluorescent protein” as usedherein is an Aequorea victoria green fluorescent protein (GFP),structural variants of GFP (i.e., circular permutants, monomericversions), folding variants of GFP (i.e., more soluble versions,superfolder versions), spectral variants of GFP (i.e., YFP, CFP), andGFP-like fluorescent proteins (i.e., DsRed and mcherry). Fluorescentproteins can be from different resources. For class Hydrozoa, GFP can befrom Aequorea victoria, Mitrocoma (synonym Halistaura), Obelia,Phialidium etc. For class Anthozoa, GFP can be from Acanthopilum,Cavernularia, Renilla, Ptilosarcus and Pennatula, Stylatula, etc. wealso have GFP-like proteins from Anemonia majna, FP595 from Anemoniasulcata, FPs from Zoanthus, etc. The term “GFP-like fluorescent protein”is used to refer to members of the Anthozoa fluorescent proteins sharingthe 11-beta strand “barrel” structure of GFP, as well as structural,folding and spectral variants thereof. The terms “GFP-likenon-fluorescent protein” and “GFP-like chromophoric protein” (or,simply, “chromophoric protein” or “chromoprotein”) are used to refer tothe Anthozoa and Hydrozoa chromophoric proteins sharing the 11-betastrand “barrel” structure of GFP, as well as structural, folding andspectral variants thereof. GFP-like proteins all share common structuraland functional characteristics, including without limitation, thecapacity to form internal chromophores without requiring accessoryco-factors, external enzymatic catalysis or substrates, other thanmolecular oxygen.

A variety of fluorescent proteins may be used in the present disclosure,including proteins that fluoresce due to intramolecular rearrangementsor the addition of cofactors that promote fluorescence. For example,green fluorescent proteins of cnidarians, which act as theirenergy-transfer acceptors in bioluminescence, are suitable fluorescentproteins for use in the fluorescent indicators. A green fluorescentprotein (“GFP”) is a protein that emits green light, and a bluefluorescent protein (“BFP”) is a protein that emits blue light. GFPshave been isolated from the Pacific Northwest jellyfish Aequoreavictoria; the sea pansy Renilla reniformis; and Phialidium gregarium(see Ward et al., (1982) Photochem. Photobiol. 35: 803-808 and Levine etal., (1982) Comp. Biochem. Physiol., 72B: 77-85). Red fluorescentprotein mCherry with the excitation wavelength at 587 nm and emissionmaxima at 610 nm. (Shaner, N. C. et. al., (2004) Nat. Biotech.)

A variety of Aequorea-related GFPs having useful excitation and emissionspectra have been engineered by modifying the amino acid sequence of anaturally occurring GFP from Aequorea victoria. See Prasher et. al.,(1992) Gene 111: 229-233; Heim et al., (1994) Proc. Natl. Acad. Sci.,USA 91: 12501-12504; U.S. Ser. No. 08/337,915, filed Nov. 10, 1994;International application PCT/US95/14692, filed Nov. 10, 1995; and U.S.Ser. No. 08/706,408, filed Aug. 30, 1996. The cDNA of GFP can beconcatenated with those encoding many other proteins; the resultingfusions often are fluorescent and retain the biochemical features of thepartner proteins. See, Cubitt et al., (1995) Trends Biochem. Sci. 20:448-455. Mutagenesis studies have produced GFP mutants with shiftedwavelengths of excitation or emission. See, Heim & Tsien (1996) CurrentBiol. 6: 178-182. Suitable pairs, for example a blue-shifted GFP mutantP4-3 (Y66H/Y145F) and an improved green mutant S65T can respectivelyserve as a donor and an acceptor for fluorescence resonance energytransfer (FRET). See, Tsien et al., (1993) Trends Cell Biol. 3: 242-245.A fluorescent protein is an Aequorea-related fluorescent protein if anycontiguous sequence of 150 amino acids of the fluorescent protein has atleast 85% sequence identity with an amino acid sequence, eithercontiguous or non-contiguous, from the wild type Aequorea greenfluorescent protein. More preferably, a fluorescent protein is anAequorea-related fluorescent protein if any contiguous sequence of 200amino acids of the fluorescent protein has at least 95% sequenceidentity with an amino acid sequence, either contiguous ornon-contiguous, from the wild type Aequorea green fluorescent protein.Similarly, the fluorescent protein can be related to Renilla orPhialidium wild-type fluorescent proteins using the same standards.

A variant of GFP with two mutations at F64L and S65 used in embodimentsof the present disclosure includes enhanced green fluorescent protein(EGFP). Its chromophore has an excitation maximum at 488 nm and emissionmaxima at 510 nm. Its fluorescent signal is significantly greater thanthat of wildtype GFP without these two mutations.

Another variant of GFP is called Cycle 3 (See, Patterson et al., (1997)Biophys. J. 73: 2782-2790, which is included herein by reference). ThisGFP variant with mutations at F99S, M153T and V163A at w.t. GFP hasimproved folding and chromophore formation at 37° C. or above.

Other fluorescent proteins can be used in the fluorescent indicators,such as, for example, yellow fluorescent protein from Vibrio fischeristrain Y-1, Peridinin-chlorophyll a binding protein from thedinoflagellate Symbiodinium sp., phycobiliproteins from marinecyanobacteria such as Synechococcus, e.g., phycoerythrin andphycocyanin, or oat phytochromes from oat reconstructed withphycoerythrobilin. These fluorescent proteins have been described inBaldwin et al., (1990) Biochemistry 29: 5509-5515, Morris et al., (1994)Plant Mol. Biol., 24: 673-677, and Wilbanks et al., (1993) J. Biol.Chem. 268: 1226-1235, and Li et al., (1995) Biochemistry 34: 7923-7930.

The term “link” as used herein refers to a physical linkage as well aslinkage that occurs by virtue of co-existence within a biologicalparticle, e.g., phage, bacteria, yeast or other eukaryotic cell.

Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will be in the formof an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used, the term “nucleotide sequence coding forexpression of” a polypeptide refers to a sequence that, upontranscription and translation of mRNA, produces the polypeptide. Thiscan include sequences containing, e.g., introns. As used herein, theterm “expression control sequences” refers to nucleic acid sequencesthat regulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalsfor introns, maintenance of the correct reading frame of that gene topermit proper translation of the mRNA, and stop codons.

Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing the fluorescent indicator codingsequence and appropriate transcriptional/translational control signals.These methods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. (See, forexample, the techniques described in Maniatis, et al., Molecular CloningA Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).

A variety of host-expression vector systems may be utilized to expressthe bioluminescent indicator coding sequence. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the calcium sensing system sequences; yeast transformed withrecombinant yeast expression vectors vectors containing the calciumsensing system sequences; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid vectors containing the calcium sensing systemsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) vectors containing the calciumsensing system sequences; or animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus vectors containing the calcium sensing system sequences,or transformed animal cell systems engineered for stable expression.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, etc. may be used in the expression vector (See, e.g.,Bitter, et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lamda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the insertedfluorescent indicator coding sequence.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for calciumsensing system.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & WileyInterscience, Ch. 13, 1988; Grant, et al., Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987,Acad. Press, N.Y., Vol. 153, pp. 516-544, 1987; Glover, DNA Cloning,Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986; and Bitter, HeterologousGene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel,Acad. Press, N.Y., Vol. 152, pp. 673-684, 1987; and The MolecularBiology of the Yeast Saccharomyces, Eds. Strathern et al., Cold SpringHarbor Press, Vols. I and II, 1982. A constitutive yeast promoter suchas ADH or LEU2 or an inducible promoter such as GAL may be used (Cloningin Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A PracticalApproach, Ed. D M Glover, IRL Press, Wash., D.C., 1986). Alternatively,vectors may be used which promote integration of foreign DNA sequencesinto the yeast chromosome.

An alternative expression system, which could be used to expressmutation assay system, is an insect system. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The calcium sensing system sequences may be clonedinto non-essential regions (for example, the polyhedrin gene) of thevirus and placed under control of an AcNPV promoter (for example thepolyhedrin promoter). Successful insertion of the calcium sensing systemsequences will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed, see Smith, et al., J. Viol. 46:584, 1983;Smith, U.S. Pat. No. 4,215,051.

DNA sequences encoding the mutation assay system of the presentdisclosure can be expressed in vitro by DNA transfer into a suitablehost cell. “Host cells” are cells in which a vector can be propagatedand its DNA expressed. The term also includes any progeny of the subjecthost cell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. However, such progeny are included when the term “hostcell” is used. Methods of stable transfer, in other words when theforeign DNA is continuously maintained in the host, are known in theart.

“Physical linkage” refers to any method known in the art forfunctionally connecting two molecules (which are termed “physicallylinked”), including without limitation, recombinant fusion with orwithout intervening domains, intein-mediated fusion, non-covalentassociation, covalent bonding (e.g., disulfide bonding and othercovalent bonding), hydrogen bonding; electrostatic bonding; andconformational bonding, e.g., antibody-antigen, and biotin-avidinassociations.

“Fused” refers to linkage by covalent bonding.

As used herein, the term “organelle” refers to cellular membrane-boundstructures such as the chloroplast, mitochondrion, and nucleus. The term“organelle” includes natural and synthetic organelles.

As used herein, the term “non-nuclear organelle” refers to any cellularmembrane bound structure present in a cell, except the nucleus.

As used herein, the term “host” or “organism” includes humans, mammals(e.g., cats, dogs, horses, etc.), living cells, and other livingorganisms. A living organism can be as simple as, for example, a singleeukaryotic cell or as complex as a mammal. Typical hosts to whichembodiments of the present disclosure may be administered will bemammals, particularly primates, especially humans. For veterinaryapplications, a wide variety of subjects will be suitable, e.g.,livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids and cell samples of the abovesubjects will be suitable for use, such as mammalian (particularlyprimate such as human) blood, urine, or tissue samples, or blood, urine,or tissue samples of the animals mentioned for veterinary applications.

“Analytes” are atoms, molecules or ions that can bind to proteins orpeptides. An analyte may bind reversibly or irreversibly and such a bondmay be covalent or non-covalent. While Ca²⁺, Ln³⁺ and Pb²⁺ are used inpreferred embodiments of this disclosure as an exemplary analyte, it isunderstood that analytes suitable with this disclosure include, but arenot limited to, metal ions including Group IIA metal ions, transitionmetal ions, and Lanthanide Series ions.

“Analytes” can also be H⁺ or OH⁻ that can bind to the proteins to changethe optical properties of the sensors. “Binding site” refers to anysection of a peptide or protein involved in forming bonds with ananalyte.

“Binding motif” is part of a binding site, often in a larger protein.The term binding site may be used interchangeably with the term bindingmotif and vice versa.

“Chemical reactions” can include the formation or dissociation of ionic,covalent, or noncovalent structures through known means. Chemicalreactions can include changes in environmental conditions such as pH,ionic strength, and temperature.

“Conformation” is the three-dimensional arrangement of the primary,secondary, and tertiary structures of a molecule, and in some instancesthe quaternary structure of a molecule, including side groups in themolecule; a change in conformation occurs when the three-dimensionalstructure of a molecule changes. A conformational change may be a shiftfrom an alpha-helix to a beta-sheet or a shift from a beta-sheet to analpha-helix.

“Detectable changes” or “responsiveness” means any response of a proteinto its microenvironment. Such detectable changes or responsiveness maybe a small change or shift in the orientation of an amino acid orpeptide fragment of the sensor polypeptide as well as, for example, achange in the primary, secondary, or tertiary structure of apolypeptide, and in some instances the quaternary structure of apolypeptide, including changes in protonation, electrical and chemicalpotential and or conformation.

A “measurable difference” in any fluorescent properties between theactive and inactive states suffices for the utility of the fluorescentprotein substrates of the disclosure in assays for activity. Ameasurable difference can be determined by measuring the amount of anyquantitative fluorescent property, e.g., the fluorescence signal at aparticular wavelength or the integral of fluorescence over the emissionspectrum.

“Operatively inserted” or “linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manners. A control sequence operativelylinked to a coding sequence is ligated such that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

“Responsive” is intended to encompass any response of a polypeptide orprotein to an interaction with an analyte.

“Fluorescence lifetime: refers to the lifetime of the fluorophoresignal, rather than its intensity. The fluorescence lifetime can bemeasured using fluorescence-lifetime imaging microscopy (FLIM), which isan imaging technique for producing an image based on the differences inthe exponential decay rate of the fluorescence from a fluorescentsample. It can be used as an imaging technique in confocal microscopy,two-photon excitation microscopy, and multiphoton tomography. Measuringfluorescence lifetime has the advantage of minimizing the effect ofphoton scattering in thick layers of sample.

Description

Analyte sensors, methods for producing and using analyte sensors,methods of detecting and/or measuring analyte activity, detecting pHchange, and/or, controlling the concentration of an analyte in a system,are disclosed. Embodiments of the analyte sensors according to thedisclosure can provide an accurate and convenient method forcharacterizing analyte activity, detecting pH change, controlling theconcentration of an analyte in a system, and the like, in both in vivoand in vitro environments, in particular in living cell imaging.

Ca²⁺ regulates numerous biological processes through spatio-temporalchanges in the cytosolic Ca²⁺ concentration and subsequent interactionswith Ca²⁺ binding proteins. The endoplasmic reticulum (ER) serves as anintracellular Ca²⁺ store and plays an essential role in cytosolic Ca²⁺homeostasis. There is a strong need to develop Ca²⁺ sensors capable ofreal-time quantitative Ca²⁺ measurements in specific subcellularenvironments without using natural Ca²⁺ binding proteins such ascalmodulin, which themselves participate as signaling molecules incells. Strategies are disclosed for creating such sensors by integratinga Ca²⁺-binding motif into chromophore sensitive locations in greenfluorescence protein. The engineered Ca²⁺ sensors exhibit largeratiometric fluorescence and absorbance changes upon Ca²⁺ binding withaffinities corresponding to the Ca²⁺ concentrations found in the ER(K_(d) values range from 0.4-2 mM). In addition to characterizing theoptical and metal binding properties of the newly developed Ca²⁺ sensorswith various spectroscopic methods, the kinetic properties were alsoexamined using stopped-flow spectrofluorimetry to ensure accuratemonitoring of dynamic Ca²⁺ changes. The developed Ca²⁺ sensor wastargeted to the ER of mammalian cell lines to monitor Ca²⁺ changesoccurring in this compartment in response to stimulation with agonists.It is contemplated that this class of Ca²⁺ sensors can be modifiedfurther to measure Ca²⁺ in other cellular compartments, providing toolsto study the contribution of these compartments to cellular Ca²⁺signaling.

An EGFP-based Ca²⁺ sensor was successfully created by grafting anEF-hand motif with a continuous Ca²⁺ binding site into wild type EGFP asscaffold protein[35]. The generated Ca²⁺ sensor (G1) exhibits a dual 510nm fluorescence intensity ratiometric change accordingly when excited at398 and 490 nm was monitored to decide the concentration of Ca²⁺.Although the dynamic range is relative small (only 10-15% change) inmammalian cell imaging, this work strongly supports the hypothesis thatGFP chromophore can be altered by introducing a Ca²⁺ inducedconformational change. The advantages of Ca²⁺ sensors by site-directedmutagenesis are listed as follows: 1) Direct design of a Ca²⁺ bindingsite on the surface of EGFP is supposed to create a bigger dynamic rangeof the signal change if its distance to chromophore is shorter than thegrafting approach. This is because the shortest distance between thesurface of GFP to the chromophore is only around 10 Å while Ca²⁺ boundto the grafted EF-hand should crosstalk to the chromophore at more than30 Å far away. This new strategy may have a more direct influence on thechromophore. 2) We chose EGFP (S65T mutant of wt.GFP) as the scaffoldprotein, as it is stable, non toxic, and exhibits robust opticalfluorescence under physiological conditions [36]. The cycle 2 mutations(M153G, V163A)[37] were created in scaffold protein to improve theprotein folding efficiency at high temperature, as poor folding willcause not only unqualify the cell imaging due to low fluorescentintensity, but also the dysfunction of the Ca²⁺-binding site. Thephysiological temperature of mammalian cell is unfavorably high, due tothe wtGFP encoded by Aequor Jellyfish inhabitance in the deep coldocean. 3) Protein with different Ca²⁺ binding affinities can be easilydeveloped by alternating the electrostatic potential of the bindingsites originated from the local negatively charged coordination ligands,according to the success of CD2-based Ca²⁺ binding protein design[38].4) The designed GFP-based Ca²⁺ sensor can specifically target variouscellular organelles or tissues by fusing different signal peptides. 5)It can overcome the limitation of currently reported Ca²⁺ sensors basedon natural Ca²⁺ binding proteins due to the perturbation of Ca²⁺signaling[39]. Furthermore, we propose to conduct nuclear magneticresonance analysis to explore the mechanism of particular moleculesinfluencing the chromophore environment and the chromophoreconformational change. This will provide solid theoretical evidences forthe development of GFP-based biosensors detecting diverse molecules.

Rationale of design Ca²⁺ binding site on the surface of GFP bysite-direct mutagenesis: FIG. 1 shows the designed Ca²⁺ sensor in EGFP(7E15.EGFP) based on following considerations: First, this Ca²⁺ bindingsite was designed to mimic that of 7E15 in CD2, formed by fivenegatively charged residues with their sidechain carboxyl oxygenorientated in a pentagonal by prymidal geometry to enabling similar Ca²⁺binding affinity. Second, tolerance of mutation for protein folding wasconsidered to avoid the perturbation of fluorescence intensity. We chosethis site according to the published paper, where three bulky aromaticresidues were mutated to be positively charged ones around this area toprevent the formation of dimerization.[40] Third, fluorescencesensitivity spots were determined by the chromophore solventaccessibility in particular location, as fluorescence tends to bequenched by exposing the chromophore to solvent. Richmond has reportedthe Cu²⁺ indicator with more than 40% fluorescent quench in response to10-100 μM Cu²⁺ by site-directed mutagenesis of residue 204 and 147[41],which demonstrated the high water accessibility around this area. Weapplied all five negatively charged residues around this area, in orderto weaken the hydrogen bonds between antiparallel beta sheets bysidechain charge repulsion. Fourth, the geological distance betweenchromophore and calcium binding site are minimized for biggestinteraction of Ca²⁺ with chromophore. Several residues such as 222 and203 involved in hydrogen network with chromophore were reported withshortest distance compacted in this region.

Embodiments of the analyte sensors according to the disclosure comprisea fluorescent host polypeptide and a molecular recognition motif thatinteracts with an analyte (e.g., calcium (or other metal as notedherein) or a flux of calcium in its microenvironment). Upon interactionof an analyte with the molecular recognition motif, the analyte sensorgenerates an optically-detectable signal (or the optically-detectablesignal is altered or the lifetime of the signal is changed) which isproduced during exposure to an analyte. The molecular recognition motifis integrated or operatively linked into (within the amino acidsequence) a fluorescent host polypeptide. The interaction of the analytewith the molecular recognition motif produces a detectable change influorescence properties (e.g., change of the intensity, or maximawavelength or the imaging of the absorption, transmitted light,fluorescent excitation or emission change, light scattering, lifetime ofthe signal, and/or energy transfer of the chromophore and the protein)of the analyte sensor based on the quantity of the analyte.

Using relevant molecular recognition motifs, the analyte sensor can beused to investigate the mechanisms of diseases, track the process ofdiseases and diagnose some diseases related to analyte activity invitro, in living cells and in vivo. In addition, a specific signalpeptide can also be useful for investigating mechanisms such as theiractivation or inhibition of diseases related to calcium (or other metalsas noted herein) activities in various cellular compartments in realtime and in situ, which is useful in biotechnology, cell biology andmedicinal chemistry, disease diagnosis and prognosis, calcium inhibitorscreening and drug development.

Embodiments of the analyte sensors include an engineered fluorescenthost polypeptide having a metal ion binding site comprising a pluralityof negatively charged residues, wherein the negatively charged residuescomprise a plurality of carboxyl oxygens orientated in a pentagonalbipyrimdal geometry wherein said geometry provides a metallic ionbinding site operatively interacting with a chromophore region of theengineered fluorescent host polypeptide such that binding of a metal ionanalyte to the molecular recognition motif modulates the emission of afluorescent signal emitted by the fluorescent host polypeptide, andoptionally, the absorbance spectrum of the engineered fluorescent hostpolypeptide.

Upon interaction of the analyte (e.g., calcium, lead, a lanthanide, andthe like) with the analyte binding site, the analyte sensor produces analtered signal relative to the analyte sensor prior to interaction. Inthis regard, the relative three dimensional position of the chromophorewithin the fluorescent host polypeptide is altered upon interaction ofthe analyte with the analyte binding site, where such alterationgenerates the altered signal.

In other words, the analyte sensors have a folding arrangement in athree-dimensional space that produces a specific signal. The analytesensor can undergo a local conformational change into another foldingarrangement with an alteration of the chromophore microenvironment underthe inducement of an analyte (e.g., calcium, lead, or a lanthanide) withthe analyte binding site. The conformational change can be detected andmeasured and compared to the signal generated by the calcium sensorprior to interaction with the analyte.

The advantages of embodiments of the present disclosure can include oneor more of the following: (i) embodiments of the present disclosure arecapable of monitoring numerous cellular events in living cells ororganisms via live cell imaging. Embodiments of the present disclosurecan provide continuous and dynamic movies of the cellular event andtheir responses by the stimuli or drugs. Embodiments of the presentdisclosure largely overcome the limitations of currently commercialavailable small molecule dyes, peptide/mimics probes with one snap shotof the analyte action; (ii) embodiments of the present disclosureinclude single fluorescent proteins that are more easily and bettertranslocated under cellular environment to probe analyte reaction insitu than FRET pairs that used two fluorescent proteins. With theaddition of signal peptides, these analyte sensors can be specificallyexpressed/placed at the cellular environments such as ER, mitochondrial,Golgi or nuclei to monitor cellular event with spatial resolution inaddition to temporal resolution. Currently available dye detectionmethods simply rely on passive diffusion of the probe through themembrane, and permits only short snapshots of calcium actions withoutthe capability of detecting reactions at targeted cellular locations.These probes do not provide continuous dynamic imaging of calciumactions due to limited cellular lifetime and specificity; (iii)embodiments of the present disclosure do not use existing/naturalcalcium binding proteins to sense metal ions (e.g., calcium, lead, or alanthanide), thus they have minimized perturbation of cellular network;(iv) embodiments of the present disclosure include single fluorescentprotein units that overcome the limitations observed with FRET-basedsensors that are prone to fluorescence photobleaching, poor orientationand translocation in the cellular compartments due to their large size;(v) the ratiometric signal change of embodiments of the presentdisclosure with absorption or excitations at 398 and 490 nm permitsquantitative and accurate measurement of the calcium (or other metal asnoted herein) action by normalizing the concentration change of thesensors and cellular and instrumental interference of the fluorescencesignal; (vi) creating different sensors with different analyteaffinities allows for monitoring of cellular response with high accuracyand sensitivity; (vii) the structural motifs used in embodiments of thepresent disclosure allow the maximal optical responses as well theoptimal molecular recognition required for chemical reactions; and(viii) the developed analyte sensors can be expressed in bacterial,mammalian cells, and animals such as mice with good optical propertiessuch as those described herein. The changes in the fluorescent andabsorbance properties of the engineered polypeptides of the disclosureinducible by metal ion binding may also be used to detect the removal ofthe metal ion resulting in a reverse change.

Thus, the systems, sensors, and methods of the present disclosure can beused to detect, measure, quantitate, and image interactions between theanalytes with the analyte binding site, in vitro and in vivo. Inparticular, embodiments of the present disclosure can be used to detect(and visualize) and/or quantitate calcium interactions or events invitro as well as in cells, tissues, and in vivo. In addition, thesystems, sensors, and methods of the present disclosure can be used todetect, measure, quantitate pH change with the analyte binding site, invitro and in vivo. Furthermore, the systems, sensors, and methods of thepresent disclosure can be used to control the concentration of ananalyte in a system.

The analyte sensors according to the disclosure can include anengineered fluorescent host polypeptide having a metal ion binding sitecomprising a plurality of negatively charged residues, wherein thenegatively charged residues comprise a plurality of carboxyl oxygensorientated in a pentagonal bipyrimdal geometry wherein said geometryprovides a metallic ion binding site operatively interacting with achromophore region of the engineered fluorescent host polypeptide suchthat binding of a metal ion analyte to the molecular recognition motifmodulates the emission of a fluorescent signal emitted by thefluorescent host polypeptide, and optionally, the absorbance spectrum ofthe engineered fluorescent host polypeptide. In an embodiment, thenegatively charged residues are on the surface of three anti-parallelbeta-sheets. In an embodiment, the negatively charged residues arespread on three strands of the protein with beta-can structure.

The native signal of the fluorescent protein is altered by the inclusionof the analyte binding site within the amino acid sequence of thefluorescent host polypeptide and the structural motif. In particular,embodiments of the present disclosure provide for insertion positions ofthe analyte binding site so that the analyte sensor produces emissionsat two or more wavelengths. In this regard, the relative threedimensional position of the chromophore within the fluorescent hostpolypeptide is altered by the inclusion of the analyte binding site andthe structural motif, where such alteration generates the alteredsignal.

Upon interaction of the analyte (e.g., calcium, lead, and/or lanthanide)with the analyte binding site, the analyte sensor produces an alteredsignal relative to the analyte sensor prior to interaction. In thisregard, the relative three dimensional position of the chromophorewithin the fluorescent host polypeptide is altered upon interaction ofthe analyte with the analyte binding site, where such alterationgenerates the altered signal. The ratiometric change of the signal(chromophore signal) after the interaction allows an accuratemeasurement of the analyte activity (e.g., in vitro and in vivo withnormalized sensor concentration). The inclusion of the structure motifallows optimal molecular recognition by incorporating essentialstructural and chemical properties required for a specific type ofanalyte. For example, inclusion of the structure motif allows for:solvent accessibility for the easy access of calcium, flexibilityrequired for the recognition, a special geometric pocket for theinteraction, a hydrophilic surface or charged environments to facilitatethe binding process and a required environment for the fast kineticrates such as good off rate required for real time measurements.

Design of calcium-binding GFP: The design of calcium binding proteins ingreen fluorescent protein was carried out using the established designprogram and the given parameters based on the pentagonal bipyramidalgeometry (Biochemistry 44: 8267-8273; J. Am. Chem. Soc. 127: 2085-2093;J. Am. Chem. Soc. 125: 6165-6171). More than 3000 potential calciumbinding sites were computationally constructed.

Several criteria were applied to rank and to choose sites: (i) any sitesthat contained mutations in the central helix (i.e. amino acids 56 to71) were removed; (ii) sites that replaced buried hydrophobic residueswith charged residues were removed to avoid folding disruptions; (iii)sites involving solvent-inaccessible residues, such as Phe8, wereeliminated since solvent accessibility is observed for many calciumbinding sites. The solvent accessibility was evaluated with the programGetArea; (iv) the mutations in the loop regions with higher flexibilitywere considered “safe” without disrupting the protein folding, whilesites involving the mutations on the β-strands were considered moreaggressive; (v) since fewer mutations are less likely to perturb thenative protein conformation, predicted sites with more existing residuesas ligands are preferred; (vi) the distance from the chromophore wasalso evaluated for the potential development of calcium sensors. Theover packing of protein was examined, and the clash with close residueswas avoided. In addition, the sites with three to four negativelycharged ligand residues were preferred based on the statistical resultsfor calcium binding proteins; and (vii) to have a potentialcalcium-induced fluorescence change, chromophore sensitive locationswere analyzed based on the dynamic and conformational properties of thefluorescent proteins.

FIG. 14 shows five calcium binding sites (termed GFP.D1, GFP.D2,GFP.D2′, GFP.D2″, and GFP.D3) located in three different positions inGFP chosen based on the criteria (Table 1). GFP.D1 is located at the endof the barrel in the loop regions. It is expected to have less effect onthe EGFP folding and structure due to the flexibility of the loopregion. GFP.D2, GFP.D2′, and GFP.D2″ are located in the loop region onthe opposite end of the barrel from GFP.D1. They contain four identicalligand residues and differ by one residue. GFP.D2 has ligands L194E,S86D, S2D, D82, and E5. L194 was mutated to be N in both GFP.D2′ andGFP.D2″. GFP.D2′ contains K85D mutation whereas GFP.D2 and GFP.D2″contains S86D. This alters the sidechain packing and electrostaticinteractions in the local environment due to the different size andcharge natures of Lys, Glu, Asn, and Ser. GFP.D3 is located in themiddle of the barrel, 14 Å to the chromophore. All ligand residues,including two natural ones and three mutations, are located on theβ-strands.

Chromophore and conformational properties of designed proteins: Fourcalcium binding sites were engineered into EGFP, and they exhibitdifferent optical properties. Among all of the bacterial-expressedproteins in E. coli, GFP.D2 is the only one that retains greenfluorescence color. As shown in FIGS. 15A and 15B, thebacterial-expressed and purified GFP.D2 and its series and wildtype EGFPexhibit absorption maxima at 490 nm. The excitation at 490 nm results inan emission maximum at 510 nm. In contrast, the rest of thebacterial-expressed proteins GFP.D1 and GFP.D3 are colorless, indicatingno chromophore formation in the bacterial-expression system. FIG. 15Bshows that the far UV CD spectra of these designed proteins have anegative maximum at 216 nm similar to EGFP, indicating that a dominantβ-sheet structure was not altered after introducing calcium bindingligand residues although the chromophore formation was perturbed.

GFP is originally from jellyfish and it was reported that a eukaryoticexpression system can facilitate chromophore formation since eukaryotecells contain machinery to aid in protein folding (J. Mol. Biol. 353:397-409). FIG. 16 shows that both GFP.D1 and GFP.D2 exhibit fluorescencewhen expressed in Hela cell. In contrast, GFP.D3 remains colorless whenexpressed in the mammalian cells, similar to its expression in bacterialsystem. These results suggest that introducing several charged residuesfor calcium binding does not affect the folding and structure of theprotein but does affect the synthesis and formation of the chromophore,which has less tolerance for environmental modifications.

Metal binding affinities and selectivity of designed GFP variants: Metalbinding capabilities for calcium and its analog lanthanide ions ofdesigned GFP variants were examined using four different methods usingbacterial expressed and purified proteins. For GFP.D2 with a correctformed chromophore, metal binding affinity was directly determined bymonitoring fluorescence signal change as a function of metalconcentration. As shown in FIG. 17A, the addition of calcium from 0 to10 mM results in a gradually decrease of fluorescent signal at 510 nmwhen excited at 398 nm. The fractional change at 510 nm can be wellfitted with the equation forming 1:1 calcium: protein complex. Thedissociate constants for calcium is 107±13. On the other hand, wildtypeEGFP does not have any significantly fluorescence signal change uponaddition of the metal ions.

Rhodamine-5N (Molecular Probes), a commercially available calciumbinding dye to was used obtain calcium and lanthanide affinity by a dyecompetition assay. As shown in FIG. 17B, Rodmine-5N shows a largefluorescence signal increase when calcium is bound in GFP.D1. In the dyecompetition assay, the solutions with constant dye and proteinconcentration were titrated with calcium until saturation was observed(FIG. 17B insertion). The binding affinities for the designed proteinswere obtained by globally fitting the spectra with themetal-and-two-ligand model. As shown in Table 1, the calcium bindingaffinities of GFP.D2 obtained by directly measurement of fluorescencesignal change are in agreement with that obtained by dye the competitionmethod.

TABLE 1 Design sites engineered into Green Fluorescent Protein. AverageCharge Average distance to of a.a. in distance Design Calcium-bindingChromophore binding binding Tb (III) Ca (II) Site Ligands (Å) sitesite-Trp (A) K_(d) (μM) K_(d) (μM) GFP.DI Q177N, 1171D, 22 −3 17 1.9 ±0.4 60 ± 5 D173, S1750, N135 GFP.D2 E5, D82, S2D, 15 −5 29 N/A 107 ± 13S86D, L194E GFP.D2′ E5, D82, K79D, 15 −4 29 32 ± 13 96 ± 7 L194N, K85DGFP.D2″ E5, 082, K79D, 15 −4 30 2.9 ± 0.3 38 ± 5 L194N, S86D GFPD3 E115,V120N, 14 −4 15 4.9 ± 0.2 57 ± 2 R122D, K113D

For 120, 177, and 194 the terbium affinities were measured in a 20 mMPIPES, 10 mM KCl, 1 mM DTT, 1% glycerol, pH 6.8. For 194, the terbiumaffinity was measured in 10 mM Tris, 1 mM DTT, 1% glycerol, pH 7.4. Thecalcium affinities for all four sites were measured in 10 mM Tris, 1 mMDTT, 1% glycerol, pH 7.4.

Calcium binding dye competition was then used to obtain calcium bindingaffinities for these bacteria expressed proteins GFP.D1, GFP. D2,GFP.D2′ and GFP.D2″ and GFP.D3. Their calcium-binding affinities are60±5, 57±2, 96±7 and 38±5 μM, respectively.

To further characterize the metal binding of the designed proteins,terbium sensitized fluorescence resonance energy transfer was used.Terbium, a calcium analog with similar ionic size and binding geometry,is intrinsically fluorescent at 545 nm and able to accept energytransferred from aromatic residues. EGFP contains 1 Trp and 10 Tyr, andthe Trp is within 30 Å of GFP.D1 and GFP.D2 and 17 Å of GFP.D3 andGFP.D4 (Table 1). As shown in FIG. 17C, the addition of terbium into theprotein results in a large increase in terbium-FRET signal at 545 nmwith excitation at 280 nm. The enhancement as a function of terbiumconcentration with the assumption of a 1:1 metal:protein complexprovided the binding affinities (Table 1). Of the three proteins testedat pH 7.4, GFP.D1 has the strongest terbium affinity (1.9±0.4 μM).GFP.D2 has a slightly weaker affinity of 4.9±0.2 μM while GFP.Ca2′exhibits a 15-foldweaker affinity of 32±13 μM. At pH 6.8, GFP.D2″exhibits a binding affinity for terbium of 2.9±0.3 μM. The addition ofcalcium and lanthanum into the terbium-protein complex significantlyreduced the fluorescence enhancement of terbium due to competition.

As shown in FIG. 17D, addition of 1 mM calcium resulted in a largedecrease in terbium fluorescence for GFP.D1, suggesting that calciumbinds to the protein and competes for terbium binding. Addition of 100μM lanthanum resulted in a fluorescence decrease to half, suggesting anestimated 5-fold lower metal binding affinity (about 10 μM). On theother hand, addition of higher concentrations of magnesium (10 mM)resulted in a relatively smaller decrease, indicating a relativelyweaker binding affinity. Similarly, GFP.D2′ exhibits a half maximaldecrease in fluorescence with 1 mM calcium or 100 μM lanthanum, which isalso more effective than magnesium. Taken together, calcium andlanthanides bind to the protein in the same pocket and have a greaterthan 20-fold selectivity over magnesium. The calcium binding sites ofthe present disclosure have calcium binding affinities with K_(d) in therange of 38-96 μM. The metal selectivity is also sufficient for theproteins to bind calcium without interference from magnesium in theextracellular environment or in the ER where calcium concentration ismuch higher than in the cytosol.

Embodiments of the present disclosure provide for analyte sensorsincluding a molecular recognition motif that binds an analyte (e.g.,calcium, lead, and/or lanthanide) and a fluorescent host polypeptide inwhich the molecular recognition motif is operatively linked to orintegrated therein. Interaction of the analyte with the molecularrecognition motif produces a detectable change. Table 2 lists someembodiments of the analyte sensors, the corresponding SEQ ID NO, andcharacteristics of the particular analyte sensor, while other analytesensors are described in SEQ ID. Nos. 115-159. Although SEQ ID NOS.1-99, and 104-105 and 115-159 includes specific order of amino acids,each of the groups (e.g., molecular recognition motif, fluorescent hostpolypeptide, and the like) could be positioned differently as long asthe analyte sensor produces results consistent with the embodimentsdisclosed herein.

TABLE 2 SEQ Amino acid ID Designator positions of Amino acid No.(Alternatives) EGFP positions Correspond to 1 EGFP-III-172 1-173,186-256 174-185 III. (Ca-G1′) 2 EGFP-E-III-172 1-173, 197-262 174-196E-III. 3 EGFP-III-F-172 1-173, 194-259 174-193 III-F. 4 EGFP-E-III-F-1721-173, 205-270 174-204 E-III-F (Ca-G1, EGFP-G1) 5 EGFP-E-III-F-172-ER25-197, 229-294 198-228 E-III-F (Ca-G1-ER) 6 EGFP-E-III-F-172-mito36-213, 245-310 214-244 E-III-F 7 EGFP-E-III-F-172-SKEAA 1-173, 206-271174-205 E-III-F 8 EGFP-E-III-F-172-D/N 1-173, 205-270 174-204 E-III-F 9EGFP-E-III-F-172-DD/NN 1-173, 205-270 174-204 E-III-F 10EGFP-E-III-F-172-L194N 1-173, 205-270 174-204 E-III-F 11 EGFP-I-1721-173, 186-251 174-185 I 12 EGFP-α-Lac1-172 1-173, 206-271 174-205α-Lac1 13 EGFP-α-Lac2-172 1-173, 206-271 174-205 α-Lac2 14EGFP-α-Lac3-172 1-173, 206-271 174-205 α-Lac3 15 EGFP-α-Lac4-172 1-173,206-271 174-205 α-Lac4 16 EGFP-III-172-C2 1-173, 186-256 174-185 III 17EGFP-E-III-172-C2 1-173, 197-262 174-196 E-III 18 EGFP-III-F-172-C21-173, 194-259 174-193 III-F 19 EGFP-E-III-F-172-C2 1-173, 205-270174-204 E-III-F (Ca-G1-37, EGFP-G1-C2) 20 EGFP-E-III-F-172-ER-C2 25-197,229-294 198-228 E-III-F 21 EGFP-E-III-F-172-mito-C2 36-213, 245-310214-244 E-III-F 22 EGFP-E-III-F-172-SKEAA-C2 1-173, 206-271 174-205E-III-F 23 EGFP-E-III-F-172-D/N-C2 1-173, 205-270 174-204 E-III-F 24EGFP-E-III-F-172-DD/NN-C2 1-173, 205-270 174-204 E-III-F 25EGFP-E-III-F-172-L194N-C2 1-173, 205-270 174-204 E-III-F 26EGFP-I-172-C2 1-173, 186-251 174-185 I 27 EGFP-α-Lac1-172-C2 1-173,206-271 174-205 α-Lac1 28 EGFP-α-Lac2-172-C2 1-173, 206-271 174-205α-Lac2 29 EGFP-α-Lac3-172-C2 1-173, 206-271 174-205 α-Lac3 30EGFP-α-Lac4-172-C2 1-173, 206-271 174-205 α-Lac4 31 EGFP-III-172-C31-173, 186-256 174-185 III 32 EGFP-E-III-172-C3 1-173, 197-262 174-196E-III 33 EGFP-III-F-172-C3 1-173, 194-259 174-193 III-F 34EGFP-E-III-F-172-C3 1-173, 205-270 174-204 E-III-F (EGFP-G1-C3) 35EGFP-E-III-F-172-ER-C3 25-197, 229-294 198-228 E-III-F 36EGFP-E-III-F-172-mito-C3 36-213, 245-310 214-244 E-III-F 37EGFP-E-III-F-172-SKEAA-C3 1-173, 206-271 174-205 E-III-F 38EGFP-E-III-F-172-D/N-C3 1-173, 205-270 174-204 E-III-F 39EGFP-E-III-F-172-DD/NN-C3 1-173, 205-270 174-204 E-III-F 40EGFP-E-III-F-172-L194N-C3 1-173, 205-270 174-204 E-III-F 41EGFP-I-172-C3 1-173, 186-251 174-185 I 42 EGFP-α-Lac1-172-C3 1-173,206-271 174-205 α-Lac1 43 EGFP-α-Lac2-172-C3 1-173, 206-271 174-205α-Lac2 44 EGFP-α-Lac3-172-C3 1-173, 206-271 174-205 α-Lac3 45EGFP-α-Lac4-172-C3 1-173, 206-271 174-205 α-Lac4 46 EGFP-III-157 1-158,171-251 159-170 III (Ca-G2′) 47 EGFP-E-III-F-157 1-158, 190-270 159-189E-III-F (Ca-G2) 48 EGFP-III-157-C2 1-158, 171-251 159-170 III 49EGFP-E-III-F-157-C2 1-158, 190-270 159-189 E-III-F 50 EGFP-III-157-C31-158, 171-251 159-170 III 51 EGFP-E-III-F-157-C3 1-158, 190-270 159-189E-III-F 52 EGFP-E-III-F-170 1-171, 203-270 172-202 E-III-F 53EGFP-E-I-F-170 1-171, 206-273 172-205 E-I-F 54 EGFP-E-III-F-170-C21-171, 203-270 172-202 E-III-F 55 EGFP-E-I-F-170-C2 1-171, 206-273172-205 E-I-F 56 EGFP-E-III-F-170-C3 1-171, 203-270 172-202 E-III-F 57EGFP-E-I-F-170-C3 1-171, 206-273 172-205 E-I-F 58 EGFP-120 1-239 16,114, 116, Ca²⁺ binding site (GFP-D3) 121, 123 59 EGFP-120b 1-239 16,112, 116, Ca²⁺ binding site 121, 123 60 EGFP-177 1-239 136, 172, 174,Ca²⁺ binding site (GFP-D1) 176, 178 61 EGFP-194a 1-239 6, 80, 83, 86,Ca²⁺ binding site (GFP-D2′) 195 62 EGFP-194b 1-239 6, 80, 83, 87, Ca²⁺binding site (GFP-D2″) 195 63 EGFP-229 1-239 79, 82, 198, 200, Ca²⁺binding site 230 64 EGFP-site1 1-239 3, 6, 83, 87, 195 Ca²⁺ binding site(GFP-D2) 65 EGFP-site1-ER 25-263  27, 30, 107, 111, Ca²⁺ binding site219 66 EGFP-site1-mito 36-274  38, 41, 118, 122, Ca²⁺ binding site 23067 EGFP-site2 1-239 16, 18, 116, 121, Ca²⁺ binding site 123 68EGFP-site3 1-239 84, 153, 155, Ca²⁺ binding site 162, 195 69 EGFP-site41-239 60, 101, 137, Ca²⁺ binding site 142, 178 70 EGFP-site5 1-239 8,13, 89, 115, Ca²⁺ binding site 120 71 EGFP-site6 1-239 8, 13, 89, 115,Ca²⁺ binding site 120 72 EGFP-120-C2 1-239 16, 114, 116, Ca²⁺ bindingsite 121, 123 73 EGFP-120b-C2 1-239 16, 112, 116, Ca²⁺ binding site 121,123 74 EGFP-177-C2 1-239 136, 172, 174, Ca²⁺ binding site 176, 178 75EGFP-194a-C2 1-239 6, 80, 83, 86, Ca²⁺ binding site 195 76 EGFP-194b-C21-239 6, 80, 83, 87, Ca²⁺ binding site 195 77 EGFP-229-C2 1-239 79, 82,198, 200, Ca²⁺ binding site 230 78 EGFP-site1-C2 1-239 3, 6, 83, 87, 195Ca²⁺ binding site 79 EGFP-site1-ER-C2 25-263  27, 30, 107, 111, Ca²⁺binding site 219 80 EGFP-site1-mito-C2 36-274  38, 41, 118, 122, Ca²⁺binding site 230 81 EGFP-site2-C2 1-239 16, 18, 116, 121, Ca²⁺ bindingsite 123 82 EGFP-site3-C2 1-239 84, 153, 155, Ca²⁺ binding site 162, 19583 EGFP-site4-C2 1-239 60, 101, 137, Ca²⁺ binding site 142, 178 84EGFP-site5-C2 1-239 8, 13, 89, 115, Ca²⁺ binding site 120 85EGFP-site6-C2 1-239 8, 13, 89, 115, Ca²⁺ binding site 120 86 EGFP-120-C31-239 16, 114, 116, Ca²⁺ binding site 121, 123 87 EGFP-120b-C3 1-239 16,112, 116, Ca²⁺ binding site 121, 123 88 EGFP-177-C3 1-239 136, 172, 174,Ca²⁺ binding site 176, 178 89 EGFP-194a-C3 1-239 6, 80, 83, 86, Ca²⁺binding site 195 90 EGFP-194b-C3 1-239 6, 80, 83, 87, Ca²⁺ binding site195 91 EGFP-229-C3 1-239 79, 82, 198, 200, Ca²⁺ binding site 230 92EGFP-site1-C3 1-239 3, 6, 83, 87, 195 Ca²⁺ binding site 93EGFP-site1-ER-C3 25-263  27, 30, 107, 111, Ca²⁺ binding site 219 94EGFP-site1-mito-C3 36-274  38, 41, 118, 122, Ca²⁺ binding site 230 95EGFP-site2-C3 1-239 16, 18, 116, 121, Ca²⁺ binding site 123 96EGFP-site3-C3 1-239 84, 153, 155, Ca²⁺ binding site 162, 195 97EGFP-site4-C3 1-239 60, 101, 137, Ca²⁺ binding site 142, 178 98EGFP-site5-C3 1-239 8, 13, 89, 115, Ca²⁺ binding site 120 99EGFP-site6-C3 1-239 8, 13, 89, 115, Ca²⁺ binding site 120

SEQ ID. No. 105 corresponds to the CaratER sensor. Residues for the ERtargeting sequence from calreticulin signal peptide is attached to theN-terminal and the ER retention sequence is attached to the C-terminal.SEQ ID. No.: 105 includes mutations for the new binding site and the ERtargeting and retention sequences at the N and C terminii, respectively.Additional sequences of the sensor are described in SEQ ID. No. 115 to159.

The fluorescent host polypeptide can have the molecular recognitionmotif inserted or integrated into the fluorescent host polypeptide atone of a number of locations, where each different insertion pointprovides an analyte sensor with different characteristics. For example,when the fluorescent host polypeptide is an enhanced fluorescent protein(EGFP), the molecular recognition motif can be inserted into thepositions 152, 172, or 170.

It should also be noted that the fluorescent host polypeptide can bemodified to enhance the thermal stability and/or the fluorescentproperties of the analyte sensor by including two or three mutations tothe fluorescent host polypeptide. In particular, the EGFP can includetwo mutations (M153T, V163A) and/or three mutations (F99S, M153T,V163A), which increase thermal stability and or fluorescence properties,as described herein. These mutations are noted in SEQ ID Nos.: 16 to 45,SEQ ID Nos.: 48 to 51, SEQ ID Nos.: 54 to 57, and SEQ ID Nos.: 72 to 99,respectively. Additional details and the examples that describe specificembodiments of the present disclosure are provided below.

Based on the fluorescence properties of the analyte sensor, a DNAconstruct of the analyte sensor may be inserted into a recombinantvector or any suitable vectors that may conveniently be subjected torecombinant DNA procedures. The specific vector can depend on the typeof host cells. For example, recombinant DNA plasmid vectors, which canexist as an extrachromosomal entity, may be a suitable vector.Alternatively, the vector may be one that, when introduced into a hostcell, is integrated into the host cell genome and replicates togetherwith the chromosome(s) into which it has been integrated. Once theanalyte sensor has been constructed, vectors comprising the fluorescentnucleic acid molecules may be formulated into a variety of compositions,such as solutions (for example, buffer solutions) to be used intransfecting host cells.

A fluorescent host polypeptide or variant thereof can be linked to themolecule directly or indirectly, using any linkage that is stable underthe conditions to which the protein-molecule complex is to be exposed.Thus, the fluorescent host polypeptide and molecule can be linked via achemical reaction between reactive groups present on the protein andmolecule, or the linkage can be mediated by a linker moiety, whichcontains reactive groups specific for the fluorescent host polypeptideand the molecule. It will be recognized that the appropriate conditionsfor linking the fluorescent host polypeptide variant and the moleculeare selected depending, for example, on the chemical nature of themolecule and the type of linkage desired. Where the molecule of interestis a polypeptide, a convenient means for linking a fluorescent hostpolypeptide variant and the molecule is by expressing them as a fusionprotein from a recombinant nucleic acid molecule, which includes apolynucleotide encoding, for example, a fluorescent host polypeptideoperatively linked to a polynucleotide encoding the polypeptidemolecule.

An embodiment of the analyte sensor may be produced as chimeric proteinsby recombinant DNA technology. Recombinant production of proteinsincluding fluorescent host polypeptides involves expressing nucleicacids having sequences that encode the proteins. Nucleic acids encodingfluorescent host polypeptides can be obtained by methods known in theart. For example, a nucleic acid encoding the protein can be isolated bya polymerase chain reaction of DNA from A. victoria using primers basedon the DNA sequence of A. victoria GFP. Mutant versions of fluorescenthost polypeptides can be made by site-specific mutagenesis of othernucleic acids encoding fluorescent proteins, or by random mutagenesiscaused by increasing the error rate of PCR of the originalpolynucleotide with 0.1 mM MnCl₂ and unbalanced nucleotideconcentrations.

The molecular recognition motif can include the analyte binding site,one or more structural motif, and a targeting motif. The analyte bindingsite and the structural can include those described above. The targetingmotif can target organelles and sub-organelles such as, but not limitedto, ER, mitochondrion, Golgi, nucleus, channels, gap junctions, andextracellular spaces. The targeting motif includes, but is not limitedto, signal peptides encoded in the proteins located in the targetorganelles. The targeting motif includes those listed in SEQ ID Nos.:5-6, 20-21, 35-36, 65-66, 79-80, 93-94, where the specific amino acidsequences are noted above. As mentioned above, the motifs can bepositioned differently than described herein as long as they havecharacteristics that are consistent with the embodiments disclosed.Additional details and the examples that describe specific embodimentsof the present disclosure are provided below.

The present disclosure provides for analyte sensors that comprise amolecular recognition motif that binds a metal ion analyte (e.g.,calcium, lead, and/or lanthanide) and a fluorescent host polypeptide inwhich the molecular recognition motif is operatively linked to orintegrated therein. Interaction of the analyte with the molecularrecognition motif produces a detectable change. The analyte sensor has aprotein sequence that includes the molecular recognition motif and thefluorescent host polypeptide selected from: SEQ ID Nos.: 5, 6, 20, 21,35, 36, 80, 81, 94, and 95.

An embodiment of the analyte sensor has at least one characteristicselected from the following: is stable at temperatures greater thanabout 30° C.; has enhanced fluorescent and optical properties(resulting, for example from mutations of the fluorescent protein (e.g.,F99S, M153T and V163A)), and combinations thereof. In particular,embodiments of the analyte sensors (denoted as C2 or C3 variants) (SEQID NOS.: 16-45, 48-51, 54-57, and 72-99) are able to maintainfluorescence in both mammalian and bacterial cells. Each of theembodiments described herein are able to bind calcium and other metalions (including but not limited to, Pb²⁺, Tb³⁺, La³⁺, and Gd³⁺).

An embodiment of the analyte sensor of the disclosure can be generatedby first constructing a molecular recognition motif that includes theanalyte binding site that is capable of responding to a metal ionanalyte and then operatively inserting the molecular recognition motifinto a fluorescent host polypeptide. Molecular recognition motifstypically have a primary structure, a secondary structure, and atertiary structure in most cases and in some cases a quaternarystructure, at least one of which can be tailored to the analyte sensorto achieve a desired level of analyte sensitivity. That is, each of theprimary structure, the secondary structure, the tertiary structure, andif present, the quaternary structure can be tailored to the analytesensor independently or in combination with one or more others of thestructures to achieve a desired level of sensitivity for the sensorrelative to the analyte. For example, the binding of the analyte to themolecular recognition motif preferably produces a change in a detectablesignal (fluorescence, for example) and the manipulation of the molecularrecognition motif manipulates the responsiveness of the sensor.

An embodiment of the analyte sensor also can allow the quantification ofan analyte due to a molecular recognition motif able to produce adetectable change upon excitation, expressing the protein, providingexcitement to the analyte sensor, and then quantifying the detectablechange. Preferably, the protein can include a fluorescent hostpolypeptide, whose emission intensity is relative to the quantity ofanalyte in a microenvironment.

One method for creating a molecular recognition motif is through the useof an integrating method. The integration method focuses on engineeringand constructing a molecular recognition motif by modifying the primary,secondary, tertiary, and/or quaternary structure of an identifiedbinding site.

An illustrative method for constructing a molecular recognition motifusing the integration method includes first identifying an analytebinding peptide that binds an analyte with specificity and thenascertaining at least a portion of a nucleic acid sequence encoding theanalyte binding peptide. Once this is accomplished, the nucleic acidsequence encoding the analyte binding peptide is tailored into amolecular recognition motif that includes an analyte binding site. Afterthe tailoring is completed, a fluorescent host polypeptide is selectedand a relevant portion of the nucleic acid sequence of the fluorescenthost polypeptide is identified, and the tailored nucleic acid sequenceencoding the analyte binding peptide is operatively linked with thefluorescent host polypeptide nucleic acid sequence into a molecularrecognition motif sequence. Finally, the molecular recognition motifsequence is expressed. In this method, the nucleic acid sequenceencoding the analyte binding peptide is tailored so as to achieve themolecular recognition motif with a desired specificity for the analyte.Preferably, the nucleic acid sequence encoding the analyte bindingpeptide is tailored to have specificity for the analyte over otheranalytes. Resultant proteins encoded by the molecular recognition motifsequence are useful products of this disclosure.

The primary structure of an analyte binding site can be selectivelymodified by inserting at least one codon into the nucleic acid sequenceencoding the analyte binding peptide. Similarly, codons for chargedamino acids can be inserted into the nucleic acid sequence encoding theanalyte binding peptide. The analyte binding site can also be modifiedby selectively manipulating and adding helices, loops, bridges orlinkers, among other methods. Charged amino acids can be inserted intothe amino acid sequence encoding the analyte binding peptide and oraromatic amino acids can be introduced into the amino acid sequenceencoding the analyte binding peptide.

Another method for generating a desired molecular recognition motif isthrough the use of a computational approach in which a computationalmethod for engineering and constructing a molecular recognition motif denovo is based on optimal binding characteristics of an analyte withother moieties. In one illustrative embodiment, using establishedcriteria for evaluating Ca²⁺ binding data, a Ca²⁺ binding site ofdesired sensitivity may be constructed by molecular modeling. Forexample, such computation algorithms may be used to develop desired ionbinding motifs based on parameters such as the metal's binding geometry,the folding of the host protein, the location of the charges on thefluorescent protein, the particular chromophores, and other criteriaspecific to the Ca²⁺ binding data.

The computational approach can be used to construct a molecularrecognition motif by accessing public and or private databases thatinclude structural data on analyte binding sites, generating at leastone preliminary analyte binding site from the structural data based oncertain previously selected criteria, selecting one or more suitableanalyte binding sites from the preliminary analyte binding sites, andconstructing the analyte binding motif by tailoring the selected analytebinding site and operatively linking it with a host protein, keeping inmind that the molecular recognition motif preferably has a specificityfor a selected analyte. The structural data typically can include aminoacid sequences, secondary structures, nucleic acid sequences, geometricparameters, electrostatic properties, and coordination properties of theanalyte binding sites, such as in protein and gene banks.

The computational approach can be performed on or by a system includingat least one database that comprises the structural data on analytebinding sites, an algorithm for generating the preliminary analytebinding sites from portions of the structural data using selectedcriteria relevant to the molecular recognition motif and rating thepreliminary analyte binding sites based on specificity for a selectedanalyte, and a computer for executing the algorithm so as to query thedatabases to generate the preliminary analyte binding sites. Thealgorithm generally is a relatively simple searching algorithm that willquery the databases based on inputted criteria.

Once the molecular recognition motif has been tailored and operativelylinked into the fluorescent host polypeptide, the analyte sensor mayshow responsiveness to analyte dependent fluorescence variations. Theresponsiveness of the analyte sensor is caused by the interaction of thefluorescent host polypeptide with the molecular recognition motif, whichthen may display fluorescence properties proportional to the analyteconcentration or flux. In particular, the responsiveness is thought tobe caused by changes in the orientation and protonation of thechromophore of the fluorescent protein. The interaction between theanalyte and the fluorescent host polypeptide may result in a shift inthe emission spectra, quantum yield, and/or extinction coefficient,which may be quantitatively analyzed in real-time to probe themicroenvironment.

In use and application, an embodiment of the analyte sensor may be usedto detect and quantify the analyte concentration and flux thereof in asample as a non-ratiometric dye. More particularly, the analyte sensoris inserted into the sample, the sample then is excited by radiation,the fluorescence from the sample then is measured using an opticaldevice, and the fluorescence or flux thereof then is analyzed toquantify or detect the analyte concentration in the sample. In order toanalyze the sample, it may be necessary to generate a standard curvebased on the fluorescence generated from known analyte concentrations.Specifically, the fluorescence signal of the analyte sensor is comparedto the fluorescence of the standard curve so as to determine theconcentration of analyte in the sample.

Fluorescent host polypeptides: The analyte sensors according to thedisclosure may comprise a fluorescent host polypeptide or polypeptide(also referred to as “optically active fluorescent host polypeptide” or“optically active fluorescent protein”). The native signal of thefluorescent protein is altered by the inclusion of the analyte bindingsite within the amino acid sequence of the fluorescent host polypeptide.Embodiments of the present disclosure provide for specific insertionpositions of the analyte binding site within the fluorescent hostpolypeptide so that the analyte sensor produces an emission that isaltered upon interaction of the analyte with the analyte binding site.In this regard, the relative three dimensional position of thechromophore within the fluorescent host polypeptide is altered by theinclusion of the analyte binding site, where such alteration generatesthe altered signal. In an embodiment, the analyte sensors can emit attwo or more distinguishable wavelengths.

Fluorescent host polypeptides suitable for use in the analyte sensors ofthe disclosure include, but are not limited to, Green FluorescentProtein isolated from Aequorea victoria (GFP), as well as a number ofGFP variants, such as enhanced fluorescent protein (EGFP). Inparticular, Aequorea green fluorescent protein (GFPs) and its enhancedfluorescent proteins have about 238 amino acid residues in a singlepolypeptide chain. The native molecule has been shown to regenerate itsintrinsic fluorescence from the totally denatured state. GFPs display astrong visible absorbance and fluorescence that is thought to begenerated by the autocyclization and oxidation of the chromophore havinga tripeptide Ser-Tyr-Gly sequence at positions 65 to 67 of the 238 aminoacid sequence. Mutations to GFPs have resulted in various shifts inabsorbance and fluorescence. The usefulness of GFPs stems fromfluorescence from GFP not requiring additional cofactors; thefluorophore is self-assembling via a cyclization reaction of the peptidebackbone.

The chromophore of GFP is formed by the cyclization of the tripeptideSer65-Tyr66-Gly67. This chromophore is located inside of the β-barrelthat is composed of 11 anti-parallel strands and a single centralα-helix. There are short helices capping the ends of the β-barrel. Thechromophore has extensive hydrogen bonding with the protein frame andcan be affected by water molecules under the different folding states.The chromophore in a tightly constructed β-barrel that exhibitsabsorption peaks at 400 and 480 nm and an emission peak at 510 nm with aquantum yield of about 0.72 when excited at 470 nm. The chromophore inenhanced green fluorescent protein (EGFP), which is GFP with a mutationS65T, has an improved fluorescence intensity and thermo-sensitivity.

Two (M153T, V163A) or three additional mutations (F99S, M153T, V163A)were added to EGFP to increase the protein expression, stability,chromophore formation at 37° C., or above.

A linker comprising specific analyte binding sites can be graftedbetween the position 170, 172, and 157, as shown in SEQ ID Nos.: 1-58,as shown in Table 2, for example.

An embodiment of the analyte binding sites can be created by mutation inthe fluorescent proteins to form a proper binding pocket without usingamino acids from a contiguous stretch of the sequence. All of thesequences shown in SEQ ID Nos.: 59-99, as shown in Table 2, for example.

Analyte Binding Site: The analyte sensor according to the disclosure canhave a molecular recognition motif that includes an analyte bindingsite. The native signal of the fluorescent protein can be altered byintegration of the analyte binding site within the amino acid sequenceof the fluorescent host polypeptide. The relative three dimensionalposition of the chromophore within the fluorescent host polypeptide maybe altered by the inclusion of the analyte binding site, where suchalteration generates the altered signal. This signal change in thesensors can results in a ratiometric change i.e. an increase, adecreases, or increases at one wavelength and an opposite change atanother wavelength at both absorption and/or fluorescence excitations.

An embodiment of the analyte binding site functions by interacting witha metal ion analyte, such interaction causing the analyte sensor toproduce an altered signal relative to the analyte sensor prior tointeraction. The relative three-dimensional position of the chromophorewithin the fluorescent host polypeptide can be altered upon interactionof the analyte with the analyte binding site, where such alterationgenerates the altered signal.

The analyte binding site can include, but is not limited to, a bindingsite where the analyte binds to the analyte sensor. The binding site canbe a location where the analyte binds to the analyte sensor. Usuallyspecific types of amino acids in specific sequential or a particularspatial arrangement may be used for a specific type of analyte.Depending on the reaction and the nature of the binding and relativealteration of the chromophore, the binding of the analyte can cause analteration in the analyte sensor signal. However, the cleavage reactionwill cause large changes of the sensor signal. This can be due to thealteration of the local environment of the three dimensional position ofthe chromophore within the fluorescent host polypeptide which results inalteration of the signal. Such alteration can be due to the perturbationof the hydrogen network, the dynamic properties, the solventaccessibility or chemical properties such as hydrophobic andelectrostatic interaction.

An embodiment of the site within the fluorescent host polypeptide forinserting the analyte binding site cleavage site preferably may beselected so that the location is accessible by a metal ion analyte. Inaddition, the location within the fluorescent host polypeptide can beselected so that the location does not substantially reduce thefluorescence from the fluorescent host polypeptide and so that thelocations do not substantially denature or alter the protein folding ofthe fluorescent host polypeptide or chromophore. Furthermore, the sitewithin the fluorescent host polypeptide for inserting the analytebinding site cleavage site can be selected based on one or more of thefollowing criteria: maximization of solvent accessibility to allowefficient enzymatic action, maximization of fluorescent/optical signalsonce the analyte binding site is operatively incorporated into thefluorescent host polypeptide; minimization of the disruption to thechromophore environment after interaction of the analyte binding sitewith the analyte; minimizing the effects on the protein folding andpacking of the fluorescent host polypeptide; and maximization of theratiometric change of chromophore signal due to interaction of theanalyte binding site with the analyte so to allow an accuratemeasurement of the analyte activity in vitro or in vivo. It should benoted that the analyte binding site can be include within or betweenmotifs of the fluorescent host polypeptide, such as within or between asecondary structure motif, a tertiary structure motif, or a quaternarystructure motif. In particular, the analyte binding site can be insertedin the loop of the β-barrel, and between loops.

Structure motifs: The inclusion of a structure motif in the molecularrecognition motif allows optimal molecular recognition by incorporatingessential structural and chemical properties required for a specifictype of analyte. For example, good solvent accessibility for easieraccess by analytes, good flexibility required for recognition, a specialgeometric pocket for the interaction, a hydrophilic surface or chargedenvironment to facilitate the binding process and a required environmentfor the fast kinetic rates such as good off rate required for real timemeasurements.

For example, but not intended to be limiting, for solvent accessibilityand flexibility such as a helix-loop-helix or partial motif can beuseful. These helix-loop-helix motifs can be from EF-hand motifs fromcalcium binding proteins such as calmodulin or trponic c, S100, or fromnucleic binding motifs, and the like. Additionally, other structuralmotifs such as beta-loop-beta or beta-loop-helix, or coiled structuresor domains and fragments that contain the cleavage sequence, and whichare located at a sensitive location relative to the chromophore with thecapability to alter the chromophore environment, can be used inembodiments of the present disclosure, as listed, for example in Table2.

Targeting Motif: A target motif may have an affinity for a target suchas a cell, a tissue, a small molecule, a protein, an organelle, asuborganelle, and the like related to a normal or pathologicalcondition, biological or physiological event of the sample or host. Thetargeting motif can have an affinity for one or more targets. Thetargeting motif can be specific or non-specific.

The non-specific targeting moiety can be selected to do one or more ofthe following: enter a cell or a cell type, enter the vasculature, enteran extracellular space, enter an intracellular space, have an affinityfor a cell surface, diffuse through a cell membrane, react with anon-specified moiety on the cell membrane, enter tumors due to leakyvasculature, and the like. The non-specific targeting moiety can includea chemical, biochemical, or biological entity that facilitates theuptake of the probe into a cell. The non-specific targeting moiety caninclude, but is not limited to, cell penetrating peptides, polyaminoacid chains, small molecules, and peptide mimics.

The purified proteins of the disclosure can also be directly injectedinto the cells or cellular space to measure the analyte concentration.Sensor proteins selected from the SEQ ID Nos. 1-99, 104-105 and 115-159can be also used to measure analyte changes in vitro such as insolution. The purified proteins can also function as a buffer orchelator to control the concentration of the analyte in vitro and invivo.

Methods of Use: It is contemplated that the analyte sensors of thedisclosure can be used in vivo and/or in vitro. The analyte sensors orsystems of the disclosure can be introduced into a cell or host, theanalyte sensors or systems can be expressed in the system, and/or theanalyte sensors or systems can be included in a transgenic animal orplant. The analyte sensor can include a specific signal peptide for thedelivery of the analyte sensor to different subcellular compartmentssuch as cytosol, nucleus, mitochondrial matrix, endoplasmic reticulum,golgi and peroxisome, and the like.

Embodiments of the present disclosure provide for methods of detectingand measuring a metal ion analyte. The methods can include: introducingan analyte sensor into a system; allowing the analyte sensor to interactwith the analyte of interest, which can interact with the analytebinding site of the analyte sensor; and detecting or measuring thefluorescent properties or changes derived from the fluorophore. As thechange in fluorescent activity of the analyte sensor is a proxy for theactivity of the analyte of interest, this method provides a means forstudying and evaluating analyte activity.

Embodiments of the method of the disclosure can include: introducing aplasmid encoding the analyte sensor into a host cell by standard genetransfer methods; expressing the analyte sensor in the host cell;allowing the analyte sensor to interact with the analyte of interest,which can interact with the analyte binding site of the analyte sensor,and thereby detect or measure a fluorescent signal or changes. As thechange in fluorescent activity of the analyte sensor is a proxy for theactivity of the analyte of interest, this method provides a means forstudying and evaluating analyte activity.

The methods can include: introducing an analyte sensor into a system;allowing the analyte sensor to interact with a metal ion analyte whichcan interact with the analyte binding site of the analyte sensor; anddetecting or measuring the fluorescent properties or changes, which canbe correlated to a pH change.

Embodiments of the present disclosure can further provide for methods ofcontrolling the concentration of one or more metal ion analytes. In anembodiment, the methods can include: introducing an analyte sensor intoa system; allowing the analyte sensor to interact with the analyte,which can interact with the analyte binding site of the analyte sensor.The bonding of the analyte with the analyte controls the amount ofanalyte in the cell or host.

Samples useful with this disclosure include biological samples,environmental samples, or any other samples for which it is desired todetermine whether a particular molecule is present therein. The samplecan be, but is not limited to, a living cell or a cell extract, whichmay be obtained from an animal or a plant. Alternatively, the cells canoriginate from or be derived from bacterial cells. Further, the cellsmay be obtained from a culture of such cells, for example, a cell line,or can be isolated from an organism. Where the method is performed usingan intact living cell or a freshly isolated tissue or organ sample, thepresence of a molecule of interest in living cells can be identified,thus providing a means to determine, for example, the intracellularcompartmentalization of the molecule in real time.

Detecting with the analyte sensor: Methods for detecting with theanalyte sensor or of a cell expressing containing an analyte sensor mayinclude, but are not limited to, illuminating the analyte sensor or cellexpressing the sensor with an illumination source such that the analytesensor or cell expressing the analyte sensor emits a radiation. Suchdetection methods may use an illumination source such as an incandescentlight source, a fluorescent light source, a halogen light source,sunlight, a laser light, and other equivalent sources. When illuminatedby such an illumination source, the analyte sensor can emit fluorescentlight that may be detected by unaided optical observation or by otherqualitative or quantitative methods. Suitable methods for measuringfluorescence of samples are known and understood by those with ordinaryskill in the art.

To overcome the limitation of slow kinetics (Zou et al., Brioche, 2007),an improvement of the off-rate constant k_(off) to 256 s⁻¹ was obtainedby redesigning the binding interface between calmodulin and itstargeting peptide in GFP-based Ca²⁺ sensors. Optimizing the protonationrate of the chromophore in GFP-based Ca²⁺ sensors will provide a meansto enhance further the accuracy with which Ca²⁺ signals can be measuredwith high temporal resolution. Ca²⁺-induced changes in CatchER's opticalproperties: The model structure of our designed Ca²⁺ sensor, CatchER,was based on the scaffold protein EGFP. The binding site is adjacent tothe chromophore (right on top of the Y66 phenolic oxygen) and next toH148, T203, and E222 (FIG. 20A); its fluorescence sensitivity may be dueto hydrogen-bond interaction. The X-ray crystal structure shows mutatedresidue sidechains protruding from the protein surface, providing accessto solvents. This putative Ca²⁺ binding site is formed by residues 147,202, 204, 223, and 225, which confer Ca²⁺-preferred geometric properties(FIG. 20B). Five variants were created by introducing charged residuesin these positions (FIGS. 20D-20H).

CatchER (D11) and its variants (D8-D10 and D12) were bacteriallyexpressed and purified using established methods (Heim & Tsien (1996)Curr. Biol. 6: 178-182; Zou et al., (2007) Biochemistry 46:12275-12288). Introducing acidic ligand residues added an absorptionmaximum at 398 nm at the expense of the 490 nm peak (FIG. 20I). ThisEGFP feature is associated with predominance of the anionic chromophore.The ratio of absorption maxima 395/488 increases from 0.2 for EGFP withno charged residue to 2.3 for D10 with four acidic residues (FIG. 20J).A fluorescence maximum of 510 nm excited at 488 nm parallels theabsorbance maxima (FIGS. 25A-25L).

Ca²⁺ binding to CatchER and its variants D9 and D10 increased absorbanceat 490 nm and decreased it at 398 nm (FIG. 25C-25E, 25M), suggestingthat Ca²⁺ binding increases the anionic chromophore. In contrast, a 510nm emission maximum increased when excited at both 395 and 488 nm (FIG.25I-25K, 25M). Among all variants, CatchER had the largest fluorescenceenhancement (about 80%) upon Ca²⁺ binding (FIG. 25K and FIG. 25M) andattained approximately 50% of EGFP fluorescence intensity. D8'sfluorescence response was negligible, possibly because it has few ligandresidues and low Ca²⁺ binding affinity.

Metal binding assisted chromophore formation, as shown by a 0.7 unitdecrease in CatchER's pK_(a) in the presence of Ca²⁺ (FIG. 26B) for avalue of 6.9, which is closer to that for EGFP. Ca²⁺ binding reverseschanges in fluorescence properties associated with adding charged ligandresidues presumably because it neutralizes the excess negative chargewhile enhancing fluorescence when excited at 488 and 395 nm. Takentogether, these results suggest a unique mechanism for CatchER,involving a concomitant recovery of fluorescence and a switch in thechromophore's ionic form.

Metal binding properties: Several lines of evidence support a simpleCatchER-Ca²⁺ stoichiometry reaction. The Job Plot suggests that Ca²⁺forms a 1:1 complex with CatchER (FIG. 26C), and the fluorescence changein response to Ca²⁺ titration can be fitted to a 1:1 binding equation(FIG. 21B). The equilibrium dialysis experiments using myoglobin(noncalcium-binding protein), EGFP (noncalcium-binding protein),CatchER, and α-lactalbumin (Ca²⁺-binding protein with K_(d)=10⁻⁹ M) withCa²⁺ demonstrate that CatchER binds Ca²⁺ with weak affinity (FIGS. 27Aand 27B).

Ca²⁺-induced chemical shift changes of several residues close to thedesigned CatchER's Ca²⁺ binding site (FIGS. 22A-22C) can also be fittedto a 1:1 binding process, with K_(d) values consistent with thosedetermined by fluorescence change. CatchER exhibits the strongest Ca²⁺binding affinity, with an apparent K_(d) of 0.18±0.02 mM, while D9 hasthe weakest, with an apparent K_(d) of 0.95±0.08 mM in 10 mM Tris pH 7.4(FIG. 20L). CatchER's dissociation constant increases to 0.48±0.07 mM inthe presence of 100 mM KCl, consistent with Ca²⁺ electrostaticinteraction. Na⁺, Cu²⁺, Zn²⁺, Mg²⁺, ATP, GTP, and GDP cannot competewith Ca²⁺ for binding CatchER (FIG. 21C), which demonstrates its goodselectivity. In vitro kinetic properties of CatchER: A stopped-flowspectrophotometer was used to record fluorescence changes upon mixing 10μM CatchER with various Ca²⁺ concentrations. Baseline corresponded toCatchER mixed with Ca²⁺-free buffer. Between 40% and 60% of the initialfluorescence increase occurred within the lag-time of the stopped-flowspectrophotometer (i.e., 2.2 ms). A plot of ΔF as a function of Ca²⁺concentration yielded a hyperbolic pattern, where the K_(d) value of0.19±0.02 mM was in reasonable agreement with the K_(d) of 0.18±0.02 mMdetermined by fluorescence equilibrium titration in the same condition(FIG. 20L). The observed rate constants were independent of the calciumconcentration between 50 and 1000 μM, with an average value of 73±16s⁻¹.

The CatchER:Ca²⁺ off-rate was measured by directly monitoring changes inthe fluorescence signal after equilibrating 10 μM CatchER with 10 μMCa²⁺ plus EGTA. About 70% of the fluorescence change was completedwithin the instrument lag-time (2.2 ms), consistent with very fast Ca²⁺release. If two half-lives would be required to complete 75% of afirst-order process of the type required for Ca²⁺ release from CatchER,a k_(off) value of ˜700 s⁻¹ can be estimated from the data in FIG. 26E).To our knowledge, CatchER exhibits the fastest off-rate of all reportedCa²⁺ sensors.

Structural analysis of Ca²⁺-CatchER interaction by high-resolution NMR:After introducing the designed Ca²⁺ binding site, residues, such asY143, T153, near binding sites or V68 around the chromophore exhibitedmore than a 1.5-ppm change, while most residues had less than a 0.4-ppmchange in Ca chemical shift between CatchER and EGFP (FIG. 29C). Thisfinding suggests that adding charged ligand residues changes localchromophore conformation, reduces fluorescence, and shifts thechromophore's ionic state toward its neutral state.

From dynamic NMR, the Ca²⁺ sensor remains monomeric in solution. Ca²⁺binding leads to significant chemical shift changes in the HSQC spectraof the T153, Y143, L42, and T43 residues, located near the designed Ca²⁺binding site (FIG. 29C). Note that the main chain of Y143 close to thedesigned site showed the largest shift. These chemical shifts werefitted to a 1:1 binding equation with a K_(d) value in agreement withthat determined by fluorescence measurements (FIG. 21B), suggesting highcorrelation between these residues. On the other hand, residues R96,Q94, F165, and V61, which protrude toward the chromophore but away fromthe designed Ca²⁺ binding site, showed no significant chemical shiftchanges, indicating that Ca²⁺ binds specifically to the designed site.

NMR can further reveal Ca²⁺-induced chromophore change, despite the lackof chromophore signal in the HSQC spectra. Q69 is buried inside theprotein and forms hydrogen bonds with the chromophore. Its singleresonance gradually becomes two with the addition of Ca²⁺ (FIG. 22B),suggesting that Ca²⁺ binding converts Q69 from a fast-exchange state totwo different slow-exchange conformations. The hydrogen bond formedbetween E222 carboxyl group and the chromophore's phenolic oxygen iscrucial to its fluorescence intensity; this residue forms a main chainhydrogen bond with L42 in the reported wild-type EGFP X-ray structure(pdb ID=1EMA). L42 also exhibits a significant Ca²⁺-induced chemicalshift change. From absorbance and fluorescence studies andhigh-resolution NMR, we can attribute the enhancement in Ca²⁺-inducedfluorescence with fast kinetics to a local conformational change closeto the designed Ca²⁺ binding site, which slows down the chemicalexchange between two chromophore ionic states (kindle fluorescence bymetal binding). Additionally, the fluorescence change via direct metalinteraction is likely to be faster than indirect interactions viaconformational changes. Ca²⁺ binding-induced fluorescence changes alsobypass the slow rate between ionic states, as we observed for G1, whichdistinguishes the sensor of the disclosure apart from GCaMP, althoughboth exhibit a similar fluorescence enhancement at 488 nm in response toCa²⁺.

Endoplasmic Reticulum Ca²⁺ concentration and release in various celltypes: CatchER was fused with the calreticulin signal peptide and KDELat the scaffold EGFP N- or C-terminus, respectively, to target it to theER (FIG. 23B). Confocal microscopy of CatchER and the ER-trackerDsRed2-ER colocalized in HEK-293 and C2C12 cells further confirmCatchER's targeting specificity to the ER (FIGS. 30A and 30B).

To determine CatchER's Ca²⁺ binding affinity, permeabilized C2C12myoblasts were exposed to increasing Ca²⁺ concentrations as described.CatchER's K_(d) was 1.07±0.26 mM in BHK cells and 1.09±0.20 mM in C2C12cells. The fluorescence intensity at the end of the experiment was fullyrecovered to the value prior to calibration, which demonstrates thatCatchER was not washed out in permeabilized BHK and C2C12 cells, furthersupporting its targeting to, and retention in the ER. The resting ERCa²⁺ concentration in HeLa, HEK293, and C2C12 cells was: 396±13.2 (n=7),742±134 (n=5), and 813±88.6 μM (n=11), respectively, in agreement withreported ER Ca²⁺ concentrations of 100-900 μM using severalCameleon-based ER sensors.

ER Ca²⁺ release evoked by ATP was measured in intact C2C12 myoblastcells (FIG. 23A), and the same batches of cell were permeabilized bydigitonin to detect IP₃-induced Ca²⁺ signaling (FIG. 23B). Fluorescencerecovered when IP₃ was washed away, and adding thapsigargin slowed thedecrease in ER Ca²⁺ concentration. Again adding IP₃ caused fluorescenceto decrease rapidly to the plateau as before, and no recovery wasobserved after washing, suggesting that thapsigargin completelyinhibited the SERCA pumps.

CatchER can detect Ca²⁺ release through the ryanodine receptor elicitedby 4-chloro-m-cresol (4-CmC) in intact cells. In contrast, nodrug-related response was observed for mCherry co-expressed in the ER(FIGS. 23C and 23D). Cytosolic Ca²⁺ was monitored in C2C12 myoblastsusing Fura-2 (FIG. 24). 4-CmC elicited a concentration-dependent SR Ca²⁺depletion, while adding 500 μM 4-CmC and 2 μM thapsigargin togetherinduced full SR Ca²⁺ depletion (FIG. 23E). CatchER reports ER Ca²⁺release in excitable and nonexcitable cells, such as HeLa and HEK 293,in response to ATP, histamine, thapsigargin, and cyclopiazonic acid(FIG. 31E-31G, 31J).

Kits: This disclosure further encompasses kits that can compromise, butare not limited to, an analyte sensor according to the disclosure,related agents that can facilitate the delivery of the protein to itsdesired destination and directions (written instructions for their use).The components listed above can be tailored to the particular biologicalevent to be monitored as described herein. A kit for use in transfectinghost cells may be assembled using the nucleic acid molecules encodingthe analyte sensor, or for labeling target polypeptides with the analytesensor. Host cell transfection kits may include at least one containercontaining one or more of the nucleic acid molecules encoding a analytesensor (or a composition including one or more of the nucleic acidmolecules or plasmids described above), which nucleic acid moleculepreferably includes plasmid. The kit can further include appropriatebuffers and reagents known in the art for administering variouscombinations of the components listed above to the host cell or hostorganism. The components of the present disclosure and carrier may beprovided in solution or in lyophilized form. When the components of thekit are in lyophilized form, the kit may optionally contain a sterileand physiologically acceptable reconstitution medium such as water,saline, buffered saline, and the like.

One aspect of the disclosure, therefore, encompasses embodiments of ananalyte sensor comprising an engineered fluorescent host polypeptidehaving a metal ion binding site comprising a plurality of negativelycharged residues, wherein the negatively charged residues comprise aplurality of carboxyl oxygens orientated in a pentagonal bipyrimdalgeometry wherein said geometry provides a metallic ion binding siteoperatively interacting with a chromophore region of the engineeredfluorescent host polypeptide such that binding of a metal ion analyte tothe molecular recognition motif modulates the emission of a fluorescentsignal emitted by the fluorescent host polypeptide, and optionally, theabsorbance spectrum of the engineered fluorescent host polypeptide.

In embodiments of this aspect of the disclosure, the negatively chargedresidues are on the surface of three anti-parallel beta-sheets.

In embodiments of this aspect of the disclosure, the negatively chargedresidues are spread on three strands of the protein with beta-canstructure.

In embodiments of this aspect of the disclosure, the amino acid sequenceof the analyte sensor can have at least 90% similarity to a sequenceselected from the group consisting of SEQ ID Nos.: 104-105 and 113-159.

In embodiments of this aspect of the disclosure, the amino acid sequenceof the analyte sensor can have at least 95% similarity to a sequenceselected from the group consisting of SEQ ID Nos.: 105.

In embodiments of this aspect of the disclosure, the amino acid sequenceof the analyte sensor is according to a sequence selected from the groupconsisting of SEQ ID Nos.: 104-105 and 113-159.

In embodiments of this aspect of the disclosure, the amino acid sequenceof the analyte sensor can have at least 90% similarity to SEQ ID No.:105.

In embodiments of this aspect of the disclosure, the amino acid sequenceof the analyte sensor can have at least 95% similarity to a sequenceselected from the group consisting of SEQ ID No.: 105.

In embodiments of this aspect of the disclosure, the amino acid sequenceof the analyte sensor is according to SEQ ID No.: 105.

In embodiments of this aspect of the disclosure, the analyte sensor canbind to a metal ion selected from the group consisting of: calcium,lead, gadolinium, lanthanum, terbium, antimony, strontium, mercury, andcadmium.

In some embodiments of this aspect of the disclosure, the analyte sensorcan binds to a metal ion selected from the group consisting of: calcium.

In embodiments of this aspect of the disclosure, the analyte sensor canfurther comprising a targeting motif for selectively targeting theendoplasmic reticulum or the sarcoplasmic reticulum of a cell.

In embodiments of this aspect of the disclosure, the analyte sensor inthe presence of an analyte bound thereto can emit a fluorescent signal,the fluorescent signal indicating binding of the analyte to the analytesensor.

In embodiments of this aspect of the disclosure, the analyte sensor inthe absence of an analyte can emit a first fluorescent signal and in thepresence of an analyte bound to the analyte sensor can emit a secondfluorescent signal, wherein the first and the second fluorescent signalsare distinguishably detectable.

In some embodiments of this aspect of the disclosure, the sensor issolubilized.

In some embodiments of this aspect of the disclosure, the sensor isattached to a solid surface.

Another aspect of the disclosure encompasses embodiments of acomposition comprising an embodiment of the analyte sensor, where thecomposition can be formulated for the detection of an analyte in a testsample.

In some embodiments of this aspect of the disclosure, the compositioncan be formulated for detection of analyte in a tissue or a cell of ananimal or human host.

In some embodiments of this aspect of the disclosure, the compositioncan be formulated for detection of analyte in an isolated cell ortissue, or in a cultured cell or tissue.

In embodiments of this aspect of the disclosure, the composition can beformulated for detection of analyte in a liquid.

In embodiments of this aspect of the disclosure, the composition canfurther comprise a pharmaceutically acceptable carrier.

Yet another aspect of the disclosure encompasses embodiments of a kitcomprising an analyte sensor according to the disclosure and packaging,the packing comprising instructions for the use of the analyte sensorfor the detection of an analyte by the analyte sensor.

Still another aspect of the disclosure encompasses embodiments of amethod for detecting an analyte, comprising: (i) providing an analytesensor according to the disclosure; (ii) providing a test samplesuspected of comprising an analyte having affinity for the molecularrecognition motif of the analyte sensor; (iii) detecting a firstfluorescent signal emitted by the analyte sensor in the absence of atest sample suspected of comprising an analyte having affinity for themolecular recognition motif of the analyte sensor; (iv) contacting theanalyte sensor with the test sample; (v) detecting a second fluorescentsignal emitted by the analyte sensor in contact with the test sample;and (vi) comparing the first fluorescent signal and the secondfluorescent signal, wherein a ratiometric change in the signal indicatesan analyte in the test sample is interacting with the analyte sensor.

Still another aspect of the disclosure encompasses embodiments of amethod for detecting an analyte, comprising: (i) providing an analytesensor according to the disclosure; (ii) providing a test samplesuspected of comprising an analyte having affinity for the molecularrecognition motif of the analyte sensor; (iii) detecting a firstabsorption signal derived from the analyte sensor in the absence of atest sample suspected of comprising an analyte having affinity for themolecular recognition motif of the analyte sensor; (iv) contacting theanalyte sensor with the test sample; (v) detecting a second absorptionsignal derived from the analyte sensor in contact with the test sample;and (vi) comparing the first absorption signal and the second absorptionsignal, wherein a ratiometric change in the absorption signal indicatesan analyte in the test sample is interacting with the analyte sensor.

Still another aspect of the disclosure encompasses embodiments of amethod for detecting an analyte, comprising: (i) providing an analytesensor according to the present disclosure; (ii) providing a test samplesuspected of comprising an analyte having affinity for the molecularrecognition motif of the analyte sensor; (iii) detecting a firstfluorescent signal emitted by the analyte sensor in the absence of atest sample suspected of comprising an analyte having affinity for themolecular recognition motif of the analyte sensor; (iv) contacting theanalyte sensor with the test sample; (v) detecting a second fluorescentsignal emitted by the analyte sensor in contact with the test sample;and (vi) comparing the first fluorescent signal and the secondfluorescent signal, wherein a ratiometric change in the lifetime of thesignal indicates an analyte in the test sample is interacting with theanalyte sensor.

In some embodiments of this aspect of the disclosure, the firstfluorescent signal in the absence of an analyte is a null emission.

In some embodiments of this aspect of the disclosure, the firstfluorescent signal and the second fluorescent signal differ inwavelength, wherein the difference in the wavelengths, and optionally inthe intensities thereof, indicates an analyte in the test sample isinteracting with the analyte sensor.

In some embodiments of this aspect of the disclosure, the firstfluorescent signal and the second fluorescent signal differ inintensity, wherein the difference in the intensities indicates ananalyte in the test sample is interacting with the analyte sensor.

In embodiments of this aspect of the disclosure, the ratiometric changein the signal intensity provides a quantitative measurement of theanalyte in the test sample.

In embodiments of this aspect of the disclosure, the ratiometric changein the signal intensity in the absorption provides a quantitativemeasurement of the analyte in the test sample.

In embodiments of this aspect of the disclosure, the changes in the lifetime signal provides a quantitative measurement of the analyte in thetest sample.

In some embodiments of this aspect of the disclosure, the analyte is ametal ion selected from the group consisting of: calcium, lead,gadolinium, lanthanum, terbium, antimony, strontium, mercury, andcadmium.

In some embodiments of this aspect of the disclosure, the test sample isa cell or tissue of an animal or human subject, or a cell or tissueisolated from an animal or human subject.

In some embodiments of this aspect of the disclosure, the method isperformed in vitro.

Another aspect of the disclosure encompasses embodiments of arecombinant nucleic acid encoding an analyte sensor according to thedisclosure.

In embodiments of this aspect of the disclosure, the recombinant nucleicacid can further comprise a vector nucleic acid sequence.

Another aspect of the disclosure encompasses embodiments of agenetically modified cell comprising a recombinant nucleic acidaccording to the disclosure.

In embodiments of this aspect of the disclosure, the cell expresses theanalyte sensor encoded by the recombinant nucleic acid.

In embodiments of this aspect of the disclosure, the analyte sensorexpressed in the cell can provide a detectable fluorescent signal,absorbance signal, and/or life time change, said signal providing aqualitative or quantitative indicator of an analyte in the cell.

Another aspect of the disclosure encompasses embodiments of a method forcharacterizing the cellular activity of an analyte comprising: (i)providing a genetically modified cell comprising a recombinant nucleicacid expressing an analyte sensor according to claim 1; (ii) expressingthe analyte sensor in the genetically modifying a cell measuring asignal produced from the analyte sensor; (iii) detecting a firstfluorescent signal emitted by the analyte sensor; (iv) detecting asecond fluorescent signal emitted by the analyte sensor after theinduction of a physiological event in the cell; and (v) comparing thefirst fluorescent signal and the second fluorescent signal, wherein aratiometric change in the signal indicates a change in the level of theanalyte in the cell associated with the physiological in cell.

Another aspect of the disclosure encompasses embodiments of a method forcharacterizing the cellular activity of an analyte comprising: (i)providing a genetically modified cell comprising a recombinant nucleicacid expressing an analyte sensor according to the present disclosure;(ii) expressing the analyte sensor in the genetically modifying a cellmeasuring a signal produced from the analyte sensor; (iii) detecting afirst absorption signal emitted by the analyte sensor; (iv) detecting asecond absorption signal emitted by the analyte sensor after theinduction of a physiological event in the cell; and (v) comparing thefirst absorption signal and the second absorption signal, wherein aratiometric change in the absorption signal indicates a change in thelevel of the analyte in the cell associated with the physiological incell.

Another aspect of the disclosure encompasses embodiments of a method forcharacterizing the cellular activity of an analyte comprising: (i)providing a genetically modified cell comprising a recombinant nucleicacid expressing an analyte sensor according to the present disclosure;(ii) expressing the analyte sensor in the genetically modifying a cellmeasuring a signal produced from the analyte sensor; (iii) detecting afirst fluorescent signal emitted by the analyte sensor; (iv) detecting asecond fluorescent signal emitted by the analyte sensor or absorbancesignal after the induction of a physiological event in the cell; and (v)comparing the first fluorescent signal and the second fluorescentsignal, wherein a ratiometric change in the lifetime of the signalindicates a change in the level of the analyte in the cell associatedwith the physiological in cell. In embodiments of this aspect of thedisclosure, the genetically modified cell is an isolated geneticallymodified cell.

In embodiments of this aspect of the disclosure, the analyte is a metalion selected from the group consisting of: calcium, lead, gadolinium,lanthanum, terbium, antimony, strontium, mercury, and cadmium.

In some embodiments of this aspect of the disclosure, the analyte iscalcium.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. In an embodiment, the term “about” can includetraditional rounding according to significant figures of the numericalvalue. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ toabout ‘y’”.

EXAMPLES Example 1

Construction of EGFP Based Ca²⁺ Sensors: The Ca²⁺ binding motifs of CaM,loop-III (DKDGNGYISAAE (SEQ ID NO.: 113) and the EF hand motifEEEIREAFRVFDKDGNGYISAAELRHVMTNL (SEQ ID NO.: 114)), were inserted intoenhanced GFP (EGFP) as previously reported (J. Biotechnol. 119: 368-378,which is incorporated herein by reference) and the insertions wereverified by automated DNA sequencing.

The cDNA encoding the EGFP variant grafted with a Ca²⁺ binding motif wascloned into bacterial and mammalian expression vectors between BamH1 andEcoR1 restriction enzyme sites. For bacterial expression, the vectorpET28(a) with a 6×His-tag was utilized. For mammalian expression, theprotein-encoding DNA was subcloned into a pcDNA3.1+ vector. The ERretention sequence, KDEL, was attached to the C-terminus and the ERtargeting sequence of calreticulin (CRsig), MLLSVPLLLGLLGLAAAD (SEQ IDNO.: 112), was attached to the N-terminus of the EGFP-based Ca²⁺ sensorsthrough PCR. The Kozak consensus sequence was placed at the N-terminusof the calreticulin sequence for the optimal initiation of proteinexpression in mammalian cells. DsRed2-ER (BD Biosciences Clontech),which contains CRsig and KDEL signal peptides at the N and C-terminals,respectively, was used as a marker for the ER in co-localizationexperiments. To improve the folding at 37° C., two additional mutations,M153T and V163A, were also added to the Ca²⁺ sensors (Nature Biotechnol.14: 315-319, Biochemistry 39: 12025-12032, each of which is incorporatedherein by reference).

Example 2

Expression and Purification of EGFP and Its Variants: EGFP and itsvariants were expressed in E. coli BL21 (DE3). Cells were grown at 37°C. in LB medium containing 30 μg/ml kanamycin to an O.D.₆₀₀ greater than0.6 before protein induction with 0.2 mM isopropyl 13-D-thiogalactoside(IPTG). Since EGFP exhibits reduced fluorescence at 37° C. in vivo,high-level expression of the soluble mature form of EGFP was achieved bygrowing the cultures overnight in LB broth at 30° C. EGFP and itsvariants were purified by sonication of the cell pellet andcentrifugation at 22,500×g for 20 min. The supernatant was injected intoa fast performance liquid chromatography (FPLC) system, AKTAprime,connected to a Hitrap Ni²⁺ chelating column (Amersham Biosciences). Theprotein was eluted from the column with a gradient of imidazole in 50 mMNaH₂PO₄/Na₂HPO₄ and 250 mM NaCl (pH 7.4) and identified by massspectrometry. Imidazole was removed by dialysis against 10 mM Tris and 1mM DTT (pH 7.4).

Ultra-violet and Visible Absorption Spectroscopy: Ultra-violet andvisible absorption spectra of EGFP and its variants were determined witha Shimadzu UV-1601 Spectrophotometer. Protein concentration wasdetermined by absorbance at 280 nm using the molar extinctioncoefficient of 21,890 M⁻¹cm⁻¹ for EGFP-wt calculated from thecontribution from aromatic residues (1 Trp and 11 Tyr) (5500 and 1490M⁻¹cm⁻¹ for Trp and Tyr, respectively). The extinction coefficients (at398 nm or 490 nm) of the EGFP variants were obtained with the Eq. (1):

$\begin{matrix}{ɛ_{P} = {ɛ_{P,{280{nm}}}\left( \frac{A_{P}}{A_{,{280\; {nm}}}} \right)}} & (1)\end{matrix}$

in which, the ε_(p) is the extinction coefficient at 398 nm or 490 nm ofEGFP variants, ε_(p,280nm) is the extinction coefficient at 280 nm ofEGFP variants, A_(p) is the absorption of EGFP variants at 398 nm or 490nm, and A_(p,280nm) is the absorption of EGFP variants at 280 nm. EGFPwas used as a reference in the measurement of the extinctioncoefficients of the EGFP variants.

Fluorescence Spectroscopy: The properties of EGFP and its variants weremonitored using a Fluorescence Spectrophotometer (Photon TechnologyInternational, Inc.) with a 10 mm path length quartz cell at 20° C.Fluorescence spectra of the chromophore in proteins were measured in theemission region of 410 to 600 nm and 500 to 600 nm with 398 and 490 nmexcitation wavelengths, respectively. The ratio of emission at 500 to600 nm when excited at 398 and 490 nm as a function of Ca²⁺concentrations was utilized to calculate the apparent dissociationconstant K_(d) for Ca²⁺ binding of various EGFP-based Ca²⁺ sensors byfitting Eq 2 with a 1:1 metal binding equation:

$\begin{matrix}{f = \frac{\left( {\lbrack P\rbrack_{r} + \lbrack{Ca}\rbrack_{r} + K_{d}} \right) - \sqrt{\left( {\lbrack P\rbrack_{r} + \lbrack{Ca}\rbrack_{r} + K_{d}} \right)^{2} - {{4\lbrack P\rbrack}_{r}\lbrack{Ca}\rbrack}_{r}}}{{2\lbrack P\rbrack}_{r}}} & (2)\end{matrix}$

in which f is the fraction of Ca²⁺ bound protein, [P]_(T) is the totalprotein concentration (mM), [Ca]_(T) is the total Ca²⁺ concentration(mM), and K_(d) is the Ca²⁺ dissociation constant of the protein. Thefraction of the protein bound with Ca²⁺ was calculated according to Eq.3:

$\begin{matrix}{f = \frac{R - R_{\min}}{R_{\max} - R_{\min}}} & (3)\end{matrix}$

in which R_(min), R, R_(max) are the fluorescence emission ratios(excited at 398 and 490 nm) or the amplitudes measured with astopped-flow spectrofluorimeter for Ca²⁺-free, Ca²⁺-bound, andCa²⁺-saturated protein, respectively. The fluorescence emission ratio(excited at 398 and 490 nm) was obtained by fitting the data to Eq. 4:

$\begin{matrix}{R = \frac{F_{({398{nm}})}}{F_{({490{nm}})}}} & (4)\end{matrix}$

in which F_((398nm)) and F_((490nm)) are the integrated fluorescenceintensities in the range of 500 to 600 nm excited at 398 and 490 nm,respectively. The dynamic range value of Ca²⁺ sensors was calculated bydividing the fluorescence emission ratio excited at 398 and 490 nm ofthe Ca²⁺ saturated state (R_(max)) with that of the Ca²⁺-free state(R_(min)).

The apparent dissociation constant for Ca²⁺ binding (K_(d)) ofEGFP-based Ca²⁺ sensors was also measured by competitive titration withRhodamine-5N. Rhodamine-5N is a fluorescent dye (Molecular Probes) witha K_(d) of 319±13 μM for Ca²⁺ in 100 mM Tris, pH 7.4. The dyeconcentration was calculated using an extinction coefficient of 63,000M⁻¹cm⁻¹ at 552 nm. Measurements with different Ca²⁺ concentrations wereperformed by maintaining the concentration of dye (10 uM) and proteinconstant. The fluorescence emission signal from 560 to 650 nm wasmeasured with a cell of 1 cm path length excited at 552 nm. The slitwidths of excitation and emission were set at 2 and 4 nm, respectively.The apparent dissociation constants were obtained by globally fittingthe spectra from 560 to 650 nm using Specfit/32 with the metal and twoligand model (Spectrum Software Associates).

The Ca²⁺ selectivity of the EGFP-based Ca²⁺ sensor was examined bymonitoring the change of the fluorescence ratio F_((398nm))/F_((490nm))with 1.0 mM Ca²⁺ in the presence of metal ions including 0.1 μM Cu²⁺,0.1 mM Zn²⁺, 10.0 mM Mg²⁺, 5.0 μM Tb³⁺, or 5.0 μM La³⁺. The normalizedchange of the ratio (AR) was calculated using Eq. 5:

$\begin{matrix}{{\Delta \; R} = {\frac{R_{metal} - R_{0}}{R_{Ca} - R_{0}} \times 100}} & (5)\end{matrix}$

in which R₀ is ratio of the EGFP-based Ca²⁺ sensor in the absence ofCa²⁺ and metal ions, R_(Ca) is the ratio of the EGFP-based Ca²⁺ sensorwith 1.0 mM Ca²⁺ in the absence of metal ions, and R_(metal) is theratio of the EGFP-based Ca²⁺ sensor with 1.0 mM Ca²⁺ in the presence ofthe metal ions. Eq (5) was also used to examine the effect of smallmolecules including adenosine triphosphate (ATP), adenosine diphosphate(ADP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), andGlutathione (GSH) on the Ca²⁺ response of GFP-based Ca²⁺ sensors. Dataare expressed as a percentage.

Stopped-flow Spectrofluorometry: Stopped-flow kinetic measurements wereperformed on a Hi-Tech SF-61 stopped-flow spectrofluorometer (10 mm pathlength, dead time of <2 ms) with a 1:1 (v/v) ratio of the protein sensorand calcium at 20° C., as described previously (J. Am. Chem. Soc. 127:2067-2074). Fluorescence emission changes associated with binding ofCa²⁺ to Ca-G1 were determined by mixing Ca²⁺ and Ca-G1 in 10 mM Tris and1 mM DTT (pH 7.4) with excitation at 398 nm and a long-pass 455 nmfilter. The concentrations of Ca²⁺ ranged from 0 to 10 mM. Fluorescenceemission changes associated with dissociation of Ca²⁺ from Ca-G1 weremeasured upon mixing Ca-G1 preloaded with Ca²⁺ in 10 mM Tris and 1 mMDTT (pH 7.4) with the same buffer. Generally, six duplicate measurementswere carried out for each point and the last three were fitted to obtainthe observed rate, k_(obs). The k_(obs) for each Ca²⁺ concentration wasobtained by fitting of the stopped-flow traces according to the singleexponential function shown in Eq. 6:

F _(t) =F ₀+Amp[1−exp(−k _(obs) t)]  (6)

in which F_(t) is the fluorescence intensity at any stopped-flow time,F₀ is the initial fluorescence intensity, Amp is the final value of thefluorescence signal at the end of the process at a given Ca²⁺concentration, k_(obs) is the observed rate of fluorescence change(s⁻¹), and t is the reaction time (s). Measurements typically differedby less than 1% between duplicate experiments.

Example 3

Cell Culture and Transfection: Both BHK-21 and HeLa cells were grown on100 mm culture dishes or glass coverslips (0.5-1.0×10⁶ cells/dish) in 35mm culture dishes in Dulbecco's Modified Eagles Medium (DMEM, SigmaChemical Co., St. Louis, Mo.) with 44 mM NaHCO₃, pH 7.2 and supplementedwith 10% (v/v) Fetal Bovine Serum (FBS), 100 U/ml penicillin and 0.1mg/ml streptomycin (Pen/Strep) at 37° C. with 5% CO₂ in a humidifiedincubation chamber. The cells were seeded and grown overnight beforetransient transfection with Ca²⁺ sensor plasmid constructs.

Plasmid DNA used for transfection was harvested from transformed E. coli(DH5□) using a QIAGEN Miniprep protocol (Qiagen). Each of the GFPvariants was individually and transiently transfected into BHK-21 andHeLa cells with Lipofectamine-2000 (Invitrogen Life Technologies) andserum-free Opti-MEMI (Gibco Invitrogen Corporation) per themanufacturer's instructions. The plasmid DNA (2 μg) with a ratio of DNAto Lipofectamine between 1:1 and 1:3 (μg/μl) was generally used in atypical transfection. Following incubation at 37° C. for 4 hrs, themedium containing the DNA-Lipofectamine complex was removed and replacedwith DMEM enriched with FBS and Pen/Strep. The cells were then grown for1 to 3 days in a humidified chamber with 5% CO₂ at 30 or 37° C. beforefluorescence or confocal microscope imaging.

Example 4

Confocal Microscope Imaging: BHK-21 and HeLa cells were transferred fromDMEM to Hank's Balanced Salt Solution without divalent cations(HBSS(--), Sigma Chemical Co., St. Louis, Mo.) media with 10 mM HEPES, 5mM NaHCO₃, 1 mM EGTA, and pH 7.2 for live imaging experiments on a LSM510 laser confocal microscope (Carl Zeiss Inc., Thornwood, N.Y.) using a100× oil-immersion objective (Zeiss, Fluar, 1.30 n.a.). Prior toimaging, cells and buffers were brought to ambient temperature andallowed to equilibrate to room air. The localization of EGFP-based Ca²⁺sensors was visualized by excitation of EGFP with the 488 nm line of anArgon laser and the narrowest bandpass filter (505-530 nm) was employedfor emission. DsRed2-ER was excited with the 543 nm line of a He—Nelaser, and emission was detected through a long-pass filter (emissionabove 560 nm). Zeiss LSM 510 software (Carl Zeiss, Inc.) was used tocontrol the image acquisition parameters. All images were acquired athigh resolution (1024×1024).

Example 5

Fluorescence Microscope Imaging and Its Quantification: BHK-21 cellswere imaged 1-3 days following transfection with GFP variants. A NikonTE200 microscope running Metafluor software (Universal Imaging) withdual excitation capability was used for the cell imaging experiments.The ratio of fluorescence emission of EGFP-based Ca²⁺ sensors (measuredat 510 nm) in response to excitation wavelengths of 385 nm and 480 nmwas measured to monitor changes in [Ca²⁺]_(ER) in time seriesexperiments. The [Ca²⁺]_(ER) in BHK-21 cells was quantified according toEq. 7:

$\begin{matrix}{\left\lbrack {Ca}^{2 +} \right\rbrack = {K_{d} \times \left( \frac{R - R_{\min}}{R_{\max} - R} \right)^{\frac{1}{n}}}} & (7)\end{matrix}$

in which R is the fluorescent emission ratio (measured at 510 nm) for385 nm/480 nm excitation at the initial state, R_(min) is the minimum ofthe emission ratio determined at the Ca²⁺-free state, R_(max) is themaximum of the emission ratio at the Ca²⁺-saturated state, K_(d) is theapparent dissociation constant (mM) and n is the Hill coefficient (n=1).Ca²⁺-free and Ca²⁺-saturated states were obtained on cells treated with5 μM ionomycin and exposed to 1.0 mM EGTA and 1.0 mM Ca²⁺, respectively.

Example 6

Design of EGFP-based Ca²⁺ Sensors with a Single Inserted Ca²⁺-bindingMotif: FIG. 1 illustrates the design of Ca²⁺ sensors made by integratinga Ca²⁺-binding motif, a combination of CaM loop-III and its flankinghelices, into EGFP based on the following criteria. First, Ca²⁺-bindingmotifs such as loop-III or intact EF-hand motif III from CaM were usedto create Ca²⁺-binding sites in EGFP. Ca²⁺ is chelated by 12-residues inthe EF-hand motif. Thus, peptide fragments of an EF-motif of CaMinteract with any CaM target enzymes, thereby producing a sensor that isunlikely to interfere with cellular signaling events. The Ca²⁺-bindingaffinity of the grafted loop can be varied by modifying charged residuesin the loop and flanking helices (J. Am. Chem. Soc. 127: 3743-3750; J.Inorg. Biochem. 99: 1376-1383, each of which are incorporated herein byreference), altering the Ca²⁺ binding affinity of any designed sensor.

Three integration sites were selected: Glu172-Asp173 within Loop-9 ofEGFP (position 1), Gln157-Lys158 within Loop-8 (position 2), andAsn144-Tyr145 within Loop-7 (position 3). Loop-III of CaM, with orwithout the flanking helices, was used as a Ca²⁺ binding motif andgrafted at these positions to construct EGFP-based Ca²⁺ sensors (FIG.1A). Next, mutations M153T and V163A were inserted into construct Ca-G1to create a sensor with improved expression at 37° C. (Ca-G1-37) (NatureBiotechnol. 14: 315-319; Biochemistry 39: 12025-12032, each of which areincorporated herein by reference). Finally, a construct with both ERtargeting sequence and retention sequence, which specifically targetsCa-G1 to the ER of mammalian cells, was designed and is referred to asCa-G1-ER.

Example 7

Spectroscopic Properties of EGFP-based Ca²⁺ Sensors and SensitiveLocations of EGFP: Spectroscopic properties of Ca²⁺ sensors were firstinvestigated using purified proteins (pH 7.4). FIGS. 2A and 2B show thevisible absorbance and the fluorescence emission spectra of EGFP-wt anddifferent Ca²⁺ sensor constructs. The spectroscopic properties includingextinction coefficients and quantum yields of Ca²⁺ sensors aresummarized in Table 3.

TABLE 3 Spectroscopic Properties of EGFP and Ca²⁺ Sensor ConstructsExtinction coefficient ε (490 nm)/ ε (398 nm)^(a) ε (490 nm) ε (398 nm)Quantum yield EGFP^(b) 9.8 55.9 5.7 0.60 Ca-G1′ 10.9 34.4 3.2 0.53 Ca-G125.9 21.5 0.8 0.59 Ca-G2′ 9.3 46.4 5.0 0.60 Ca-G2 8.5 38.6 4.5 0.69Ca-G3′^(c) N/A^(d) N/A N/A N/A ^(a)□ is the extinction coefficient inunits of 10³ M⁻¹ cm⁻¹. The wavelengths in absorption peaks are shown inthe parentheses. ^(b)EGFP-wt was used as a reference in the calculationof absorbance extinction coefficient (ε) and fluorescent quantum yieldof EGFP variants. ^(c)The chromophore was not formed in Ca-G3′. ^(d)N/A,not available.

The insertion of loop-III of CaM at Gln157-Lys158 of EGFP (Ca-G2′ andCa-G2 (only Ca-G2′ is shown in FIGS. 2A and 2B), FIG. 1A) resulted in aprotein with spectroscopic properties similar to EGFP-wt with a slightdecrease in absorbance intensity. Note that the major absorbance peak at490 nm and minor absorbance peak at 398 nm reflect the relativepopulation of anionic and neutral states of the chromophore. FIG. 2Bshows that excitation at 398 nm (the neutral state) contributed greatlyto the emission peak at 510 nm.

As shown in Table 3, the constructs with a Ca²⁺-binding motif grafted atGln157-Lys158 (position 2) (Ca-G2′ and Ca-G2) had spectroscopicproperties (extinction coefficients and quantum yield constants at both398 nm and 490 nm) similar to that of EGFP-wt. The integrating loop IIIof CaM at Glu172-Asp173 of EGFP (Ca-G1′) resulted in the formation of aprotein which showed a slight increase of absorbance at 398 nm and adecrease of absorbance at 490 nm compared to EGFP-wt. Moreover, theinsertion of loop III containing the flanking EF-helices at the samelocation (Ca-G1) resulted in a protein which had a further increase inabsorbance at 398 nm and a decrease at 490 nm. The extinctioncoefficients of Ca-G1 were increased 2.6-fold at 398 nm and decreasedabout 60% at 490 nm compared to EGFP-wt. Concurrently, a correspondingincrease in fluorescence emission was observed for both Ca-G1′ and Ca-G1(FIG. 2B).

In contrast, the chromophore was not formed after insertion of loop IIIat Asn144-Tyr145 of EGFP (Ca-G3′), indicated by the lack of greenfluorescence in the bacterial expression as well as in the purifiedprotein. Thus, the integration of a Ca²⁺ binding motif at Glu172-Asp173in EGFP significantly shifts the population of the chromophore from theanionic state as indicated by the 490 nm peak to the neutral state asindicated by 398 nm peak. It is likely that Glu172-Asp173 of EGFP is achromophore sensitive location.

CD analysis was performed to test whether the changes in the chromophoreproperties of the Ca²⁺ sensor constructs were due to structural changes.All Ca²⁺ sensor constructs exhibited CD spectra similar to that ofEGFP-wt (FIG. 7), suggesting that the insertion of a Ca²⁺ binding motifinto EGFP did not significantly change the folding of the β-sheetstructure of GFP.

The pH sensitivity of the optical properties of Ca-G1′, since a fewGFP-based biosensors have been reported to be pH sensitive. FIGS. 8A and8B shows the absorbance spectra of Ca-G1′ as a function of pH. ChangingpH from 9.0 to 5.0 resulted in an increase of the absorbance at 398 nmand a decrease of the absorbance at 488 nm. The pK_(a) of Ca-G1′ is7.45±0.05 whereas the pK_(a) of EGFP is 6.0. These data suggest that theoptical properties of the designed Ca²⁺ sensor are more sensitive to pHat physiological pH than those of EGFP-wt.

Example 8

Effect of Ca²⁺ Binding on Spectroscopic Properties of EGFP-based Ca²⁺Sensors: As shown in FIG. 3A, an increase in absorbance at 398 nmconcomitant with a decrease at 490 nm was observed in response to theaddition of Ca²⁺ to Ca-G1-37. Similarly, Ca²⁺ binding resulted in anincrease in fluorescence with excitation at 398 nm (FIG. 3B) and adecrease with excitation at 490 nm (FIG. 3C).

The dynamic range value was 1.8 and was calculated by dividing thefluorescence emission ratio excited at 398 and 490 nm of the Ca²⁺saturated state (R_(max)) by that of the Ca²⁺-free state (R_(min)) (seeExperimental procedures). FIG. 3D shows the fluorescence emission ratio,F_((398nm))/F_((490nm)), of Ca-G1-37 as a function of Ca²⁺concentration. The normalized fluorescence emission ratio change couldbe fitted as a 1:1 Ca-G1-37-Ca²⁺ complex (Eq 2), yielding an apparentdissociation constant (K_(d)=0.44±0.04 mM) for its Ca²⁺ bindingaffinity. The Ca²⁺ binding affinity of EGFP-based Ca²⁺ sensors was alsodetermined using a Rhodamine-5N competition titration approach. The Ca²⁺binding affinities of these Ca²⁺ sensors varied from 0.4 to 2 mM (Table4), as determined by different techniques.

TABLE 4 Comparison of Ca²⁺ Binding Affinities of Different EGFP-basedCa²⁺ Sensors Ca²⁺ Binding Affinity, K_(d) (mM) Rhodamine-5N Ca²⁺titration Competitive titration Ca-G1′ 2.0 ± 0.4  0.9 ± 0.2 Ca-G1 0.8 ±0.1^(a) 0.4 ± 0.1 0.8 ± 0.1^(b) 0.6 ± 0.1^(c) Ca-G1-37 0.44 ± 0.04  0.2± 0.1 Ca-G2′ N/A 0.8 ± 0.2 Ca-G2 N/A 0.2 ± 0.1 Ca-G3′ N/A 0.7 ± 0.2^(a)estimated with results of fluorescence spectrophotometer.^(b)estimated with fitting Scheme 1 using results from stopped-flowspectrofluorometer. ^(c)estimated with fitting normalized changes (Amp)of stopped-flow spectrofluorimeter.

These values agreed with the approximate calcium concentration found incellular compartments such as the ER, making these Ca²⁺ sensorspromising candidates for physiological experiments in living cells.

Example 9

Ca²⁺ Selectivity of the EGFP-based Ca²⁺ Sensor: The binding selectivityof the developed Ca²⁺ sensors for Ca²⁺ was examined by measuring thechange of the ratio F_((398nm))/F_((490nm)) in the presence of 1.0 mMCa²⁺ before and following the addition of various metal ions. In cells,total metal concentrations for Cu²⁺, Zn²⁺, and Mg²⁺ are estimated to beapproximately 10 μM, approximately 0.1 mM, and greater than 10 mM,respectively. However, the free levels of these metal ions aresignificantly lower than the total concentrations, which protects thecell against potentially toxic reactions. For example, intracellularfree copper is not detected and copper chaperone is used in vivo toallocate copper to its target proteins directly.

FIG. 4A shows the Ca²⁺ responses of sensor Ca-G1-37 in the presence ofCu²⁺, Zn²⁺, Mg²⁺, Tb³⁺, and La³⁺. Note that no effect of Cu²⁺ (0.1 μM)on the fluorescence response of the sensor for Ca²⁺ was observed(101.95±3.02% compared to the reference value of 100% for 1.0 mM Ca²⁺).Zn²⁺ (0.1 mM) and Mg²⁺ (10.0 mM) produced only a small change in thefluorescence response (reduction to 85.71±3.34% and 74.29±1.22%,respectively). Non-physiological metal ions, such as Tb³⁺ (5.0 μM) andLa³⁺ (5.0 μM) have metal coordination properties similar to Ca²⁺ and areable to compete more strongly with Ca²⁺ responses of the sensor(0.15±5.4% and 16.0±9.0%, respectively). These results suggest that thedeveloped Ca²⁺ sensor, Ca-G1-37, has good metal selectivity for Ca²⁺,La³⁺ and Tb³⁺ and only to a lesser degree with the other physiologicalmetal ions.

The effects of small molecules including adenosine triphosphate (ATP),adenosine diphosphate (ADP), guanosine triphosphate (GTP), guanosinediphosphate (GDP), and Glutathione (GSH) on the Ca²⁺ response ofGFP-based Ca²⁺ sensors were also analyzed by measuring the change of theratio F_((398nm))/F_((490nm)) in the presence of 1.0 mM Ca²⁺ before andfollowing their addition.

FIG. 4B indicates that the addition of ATP (0.2 mM), ADP (0.2 mM), GTP(0.1 mM), GDP (0.1 mM), and GSH (1.0 mM) only resulted in a smalldecrease in the fluorescence response (reduction to 85.75±13.98%,96.17±1.36%, 88.30±8.09%, 93.29±1.01%, and 89.18±2.90%, respectively).These results indicate that the developed Ca²⁺ sensor, Ca-G1-37, has ahigh Ca²⁺ binding affinity to compete with small molecules includingATP, ADP, GTP, GDP, and GSH in the intracellular environment.

Example 10

Kinetics of Ca²⁺ Binding to the EGFP-based Ca²⁺ Sensor: As shown in FIG.5A, mixing Ca-G1 with various concentrations of Ca²⁺ resulted in a rapidincrease in the fluorescence emission at 510 nm with excitation at 398nm. The change in fluorescence signal is consistent with a singleexponential function (Eq. 6) yielding observed rates for fluorescenceemission change (k_(obs)) and amplitudes (Amp).

As shown in FIG. 5B, the k_(obs) values of Ca-G1 decreased withincreasing concentration of Ca²⁺, consistent with the kinetic model ofScheme 1, in which Ca²⁺ rapidly associates with one species of Ca-G1that is in equilibrium with a second form of the biosensor. Theincreases in fluorescence emission excited at 398 nm of Ca-G1 observedupon Ca²⁺ binding as shown in FIG. 5A further suggest that the neutralform of Ca-G1 is the species that binds Ca²⁺ (E** in Scheme 1), whereasthe anionic form of the biosensor (E*) does not bind Ca²⁺.

According to this kinetic model, k₁ is the first order rate constant(s⁻¹) for the conversion of the anionic species to the neutral speciesof Ca-G1, k₂ is first order rate constant (s⁻¹) for the conversion ofthe neutral species to the anionic form of Ca-G1, and K_(d2) representsthe apparent dissociation constant for the binding of Ca²⁺ to theneutral form of Ca-G1 (mM).

By fitting k_(obs) values determined as a function of Ca²⁺ concentrationto Eq 8, the k₁ and k₂ values were estimated to be 9.5±0.3 s⁻¹ and14.0±0.6 s⁻¹, respectively, and a K_(d2) value of 0.8±0.1 mM wasdetermined. The K_(d2) value was independently estimated to be 0.6±0.1mM by fitting the normalized amplitude in fluorescence emission as afunction of the concentration of Ca²⁺ by using Eq. 2 (FIG. 5C). Withinerrors associated with the measurements, the K_(d) values determinedusing stopped-flow fluorescence spectroscopy are in agreement with theK_(d) value independently determined in static titrations using aspectrofluorometer, which yielded a K_(d) value of 0.8±0.1 mM (Table 1).This, in turn, strongly supports the validity of the proposed minimalkinetic mechanism of Scheme 1 for Ca²⁺ binding to Ca-G1, where rates offluorescence changes associated with Ca²⁺ binding to the neutral speciesof Ca-G1 reflect rates of interconversion of the neutral and anionicforms of Ca-G1, as compared to the rapid association and dissociation ofCa²⁺ to and from the biosensor.

$\begin{matrix}{k_{obs} = {k_{1} + {k_{2}\left( \frac{K_{d\; 2}}{K_{d\; 2} + \left\lbrack {Ca}^{2 +} \right\rbrack} \right)}}} & (8)\end{matrix}$

According to the minimal kinetic mechanism of Scheme 1 and the datashown in FIG. 5A, the release of Ca²⁺ from preloaded Ca-G1 is expectedto be associated with a decrease in fluorescence whose rate offluorescence change represents the slow rate of conversion from theneutral to the anionic form of Ca-G1, i.e., k₂. Consequently,stopped-flow spectroscopy was utilized to independently determine k₂ bymixing equal volumes of Ca²⁺-saturated sensor with 10 mM Tris and 1 mMDTT (pH 7.4). As expected, the fluorescence intensity at 510 nmdecreased following Ca²⁺ release and the time course of fluorescencechange was consistent with a single exponential process (Eq. 6).

As shown in FIG. 5D, a k_(obs) value of 16.9±1.0 s⁻¹ was estimated inthis experiment by fitting the data to Eq. 6, in good agreement with thevalue of 14 s⁻¹ determined for k₂ from the data in FIG. 5B. Together,the kinetic data support the conclusion that Ca²⁺ rapidly associateswith and dissociates from the neutral form of Ca-G1, yielding a changein the relative amounts of neutral and anionic species that isassociated with a change in the intensity of the fluorescence signalfrom Ca-G1.

Ca²⁺ binding to Ca-G1 results in a rapid shift of the chemicalequilibrium of the chromophore between its anionic and neutral states(Scheme 1). This conclusion is supported by visible absorption,fluorescence emission, and stopped-flow fluorescence data. Both kineticand thermodynamic parameters, including the forward and reverse rateconstants for the interconversion of the anionic and neutral states ofthe chromophore, as well as the apparent dissociation constant forbinding of Ca²⁺ to Ca-G1 were determined using stopped-flow fluorescencemeasurements. This approach established that the rates of Ca²⁺association and dissociation to and from the sensor must besignificantly larger than both the forward and reverse first-order rateconstants that define the chemical equilibrium of the chromophore (k₁and k₂ in Scheme 1), which are between ˜10 and ˜20 s⁻¹. The rate of Ca²⁺association to proteins is generally a diffusion-limited process with anon-rate (k_(m)) equal or greater than 1×10⁶ M⁻¹s⁻¹. Since the apparentdissociation constant for the Ca²⁺ binding process determined in thisstudy for Ca-G1 is ˜0.8 mM for Ca-G1, an off-rate (k_(off)) of ˜800 s⁻¹can be estimated from k_(off)=k_(on)×K_(d). Whereas the on-rate ofGFP-based Ca²⁺ sensors is generally not the limiting factor in Ca²⁺measurements, the slow off-rate exhibited by Ca²⁺ sensors limits theirusefulness in monitoring changes in Ca²⁺ concentration in vivo,especially for fast Ca²⁺ oscillations. To overcome this limitation, animprovement of the off-rate constant k_(off) to 256 s⁻¹ was obtained byredesigning the binding interface between calmodulin and its targetingpeptide in GFP-based Ca²⁺ sensors. Optimizing the protonation rate ofthe chromophore in GFP-based Ca²⁺ sensors will provide a means toenhance further the accuracy with which Ca²⁺ signals can be measuredwith high temporal resolution.

Example 11

Monitoring ER Ca²⁺ Responses in Cells: Localization of the Ca²⁺ sensor,Ca-G1-ER, was confirmed in HeLa cells by co-transfecting the cells withthe ER marker DsRed2-ER that has been shown to localize exclusively tothis region in mammalian cells. FIG. 6 shows images taken through thegreen (A, Ca-G1-ER) and red (B, DsRed2-ER) channels which were excitedat 488 and 543 nm, respectively. The merged image (FIG. 6C) indicatesthe complete co-localization of Ca-G1-ER with the ER marker DsRed2-ER inthe ER of HeLa cells. FIG. 6D shows the ER distribution of Ca-G1-ER in aBHK-21 cell, a mammalian fibroblast cell line. Note the same granulardistribution of Ca-G1-ER in FIGS. 6A and 6D, suggesting that the Ca²⁺sensor also specifically localizes to the ER of BHK cells. In contrast,Ca-G1, which lacked the ER signal peptides, was expressed diffuselythroughout the cytoplasm of the cells, thereby serving as a negativecontrol (data not shown).

BHK-21 cells have been used previously to investigate the physiologicalroles of [Ca²⁺]_(ER) in intact cells by using small, low-affinity Ca²⁺indicators. ATP (100 μM) is a Ca²⁺-mobilizing agonist of this cell typeand elicits Ca²⁺ release from the ER through Ins(1,4,5)P₃-mediatedpathways. As shown in FIG. 6E, the addition of ATP (100 μM) resulted ina significant decrease (7.3±0.6% relative change) in the fluorescenceratio measured at 510 nm (excitation at 385 and 480 nm). The experimentshows five representative cells imaged in the same experiment and theresults are typical of results obtained in 5 independent experiments.This decrease of [Ca²⁺]_(ER) was also observed following application ofATP in Ca²⁺-free buffer, suggesting that ATP released Ca²⁺ from the ERindependent of extracellular Ca²⁺. The refilling of the Ca²⁺ storerequired several minutes in the presence of normal extracellular Ca²⁺ inthe medium. Similarly, the addition of the Ca²⁺ ionophore, ionomycin,under Ca²⁺ free conditions significantly emptied the ER store asindicated by the decreased 385 nm/480 nm fluorescence ratio. To obtainan estimate of [Ca²⁺]_(ER), a pseudo-calibration was performed in BHK-21cells using Eq. 7 and a K_(d) of 0.8 mM for Ca-G1 as shown in Table 1(FIG. 6F). The 385 nm/480 nm fluorescence ratio decreased to a minimumvalue (R_(min)) following a wash with Ca²⁺ free medium (EGTA) and thesubsequent addition of ionomycin (approximately 5 μM) to the Ca²⁺-freemedium (estimated to contain less than 10 nM Ca²⁺ using the freewareprogram ‘Bound and Determined’). The addition of millimolarextracellular Ca²⁺ (approximately 1 mM) resulted in a large increase inthe Ca²⁺ signal with a plateau that approached the saturation state witha maximum of 385 nm:480 nm fluorescence ratio (R_(max)) The initial Ca²⁺concentration in the ER of the BHK-21 cell was estimated to be less than1 mM using Eq 7 and addition of ATP (100 μM) reduced [Ca²⁺]_(ER) toapproximately 0.15 mM (FIG. 6F). As expected, no significantfluorescence signal change was observed in response to the aboveexperimental protocol in cells transfected with the wild type controlconstruct, EGFP-wt-ER (data not shown). These imaging experimentsdemonstrate the usefulness of this novel class of Ca²⁺ sensors in livingcells and it is anticipated that their future application willfacilitate the investigation of the role of the ER in Ca²⁺ signaling andCa²⁺ homeostasis.

Example 12

Variant Constructs: The GFP variant EGFP-D2 (SEQ ID No.: 64) with adiscontinuous calcium binding site (S2D, S86D, L194E), cycle 3 (F99S,M153T, V163A) mutations was made through site-directed mutagenesis withPCR and turbo pfu (Strategene) following the manufacturer's suggestionswith EGFP (S65T, F64L, V22L, M218I, H231L) as the initial template.

EGFP-G1 contains a continuous EF-hand Ca²⁺ binding motif III that wasinserted by several rounds of PCR utilizing turbo pfu. The linear DNAwas ligated with T4 DNA ligase (Promega) following the manufacturer'sinstructions, and the circular DNA was transformed into E. coli DH5αcompetent cells for DNA amplification. The variant DNA was verified byautomated sequencing. The cDNA encoding the EGFP variants with BamH Iand EcoR I restriction enzyme sites between the N and C terminals weresubcloned into mammalian expression vector pcDNA3.1+ that uses the CMVpromoter.

Example 13

Bacterial Expression and Purification: The proteins were expressed fromthe vector pet28a (EMD Biosciences) with a 6×His-tag using E. coliBL21(DE3) in LB-kanamycin (30 μg/mL). Expression was induced at anO.D₆₀₀ of 0.6 with 0.2 mM IPTG and expression was allowed to continuefor 21 hrs before the cells were harvested by centrifugation. For thesestudies, the temperature was controlled at both 30° C. and 37° C. afterinduction. The expression of EGFP and its variants was monitored withthe fluorescence intensity at 510 nm with a Fluo-star instrument and anexcitation wavelength of 488 nm.

Protein purification was with an Amersham-Pharmacia 5 mL HiTrapchelating HP column charged with nickel. The cell pellets wereresuspended in 20 mM Tris, 10 mM NaCl, 0.1% Triton X-100, pH 8.8 andsonicated. The cellular debris was removed by centrifugation and theprotein was loaded onto the prepared HiTrap column connected to anAmersham-Pharmacia AktaPrime FPLC. After washing to remove contaminantproteins, the protein of interest was eluted with an imidazole gradient.Contaminant imidazole was removed by dialysis, and the protein wasfurther purified using a HiTrap Q ion-exchange column (Amersham) with aNaCl gradient at pH 8.0. Protein purity was verified by SDS-PAGE.

Example 14

Mammalian Cell Culture: HeLa cells were grown on 60 mm culture dishes inDulbecco's Modified Eagles Medium (DMEM, Sigma Chemical Co., St. Louis,Mo.) with 44 mM NaHCO₃, pH 7.2, and supplemented with 10% (v/v) fetalbovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin(Pen/Strep) at 37° C. with 5% CO₂ humidified incubation chamber. HeLacells were grown to confluency before transient transfection.

Plasmid DNA used for transfection was harvested from transformed E. coli(DH5α) using QIAGEN's miniprep protocol (Qiagen). Each of the nine GFPvariants were individually and transiently transfected into HeLa cellswith Liptofectamine-2000 (Invitrogen Life Technologies) and serum-freeOpti-MEMI (Gibco Invitrogen Corporation) as per the manufacturer'sinstructions. A typical transfection consisted of 1 or 2 μg plasmid DNAwith a ratio of DNA to Liptofectamine between 1:1 and 1:3 (μg/μl)dependent upon the protein construct. Protein expression was allowed toproceed for 48 and 72 h before inverted epifluorescence imaging. Controltransfections with EGFP were performed in the same conditions as eachconstruct.

Example 15

Measurement of fluorescent intensity: Three 1 ml samples were collectedat time points throughout the expression, and centrifuged at 14 K rpmfor 3 min. The cell pellets were resuspended in 1 ml of Tris buffer atpH 7.4, and 200 μl was analyzed using a FLUOstar OPTIMA (BMG Labtech)plate reader with excitation filters of 390 and/or 460 nm and anemission filter at 510 nm.

Example 16

Fluorescence microscopy/imaging and its quantifications: An invertedepifluorescence microscope (Zeiss Axiovert 200) was utilized forfluorescence intensity screening in vivo. The microscope was equippedwith a xenon arc Lamp, filters for Sapphire GFP with 398 nm excitationand 510 nm emission, with standard DAPI, FITC, and Texas Red filters,and transmitted light. An Axiocam 5 CCD camera was connected to themicroscope at a right angle to the stage, and Zeiss Axiovision Rel 4.3software was used for data collection and analysis. For fluorescenceintensity measurements a 40× dry objective was used with Sapphire GFPand FITC filters and exposure times from 50 to 2000 ms. The images withexposure allowing for fluorescence intensity within the dynamic rangewere utilized for data analysis. The fluorescence intensity measured inthis time range was a linear function of the exposure time.

Both the area and mean fluorescence intensity of transfected cells (n>20cells per image) were measured and the total mean fluorescence intensityof cells in each imaged field was obtained with the calculation of Eq.(9):

$\begin{matrix}{F = \frac{\sum\limits_{i = 1}^{n}{S_{i}F_{i}}}{\sum\limits_{i = 1}^{n}S_{i}}} & (9)\end{matrix}$

in which, F is the total mean fluorescence intensity excited at 398 nmor 480 nm of cells in each image, and n is the number of fluorescentcells. S_(i) is the area of i^(th) fluorescent cell and F_(i) is the meafluorescent intensity excited at 398 nm or 480 nm of i^(th) fluorescentcell.

The total mea fluorescent intensity excited at 398 nm or 480 nm of theHeLa cells three days after transfection with EGFP-G1-C3 was used as areference, and the fluorescence intensity excited at differentwavelengths of the HeLa cells grown for different times with other GFPvariants was expressed as a percentage of EGFP-G1-C3 fluorescenceaccording to Eq. (10):

$\begin{matrix}{F^{\prime} = {\frac{F}{F_{0}} \times 100}} & (10)\end{matrix}$

in which, the F′ is the relative fluorescent intensity excited at 398 nmor 480 nm of the HeLa cells, F is the total mean fluorescence intensityexcited at 398 nm or 480 nm of the HeLa cells, and F₀ is the total meafluorescent intensity excited at 398 nm or 480 nm of the HeLa cellsincubated for three days after transfection with EGFP-G1-C3.

Example 17

Measurement of ultra-violet (UV) and visible absorption spectrum:Spectroscopic properties of EGFP and its variants were measured by UVand visible absorption spectra with a Shimadzu UV and Visible LightSpectrophotometer from 600 to 220 nm. The concentrations of the proteinswere determined by UV-vis absorbance at 280 nm using the molarextinction coefficient of 21,890 M⁻¹cm⁻¹ calculated from thecontribution from aromatic residues (1 Trp and 11 Tyr) (5500 and 1490M⁻¹cm⁻¹ for Trp and Tyr, respectively). The extinction coefficient at398 nm or 490 nm of the EGFP variants were obtained with the Eq. (11):

$\begin{matrix}{ɛ_{P} = {ɛ_{P,{280\; {nm}}}\left( \frac{A_{P}}{A_{P,{280\; {nm}}}} \right)}} & (11)\end{matrix}$

in which, ε_(p) is the extinction coefficient at 398 nm or 490 nm ofEGFP variants, ε_(p,280nm) is the extinction coefficient at 280 nm ofEGFP variants, A_(p) is the absorption of EGFP variants at 398 nm or 490nm, and A_(p,280nm) is the absorption of EGFP variants at 280 nm. EGFPwas used as a reference in the measurement of the extinctioncoefficients of the variants.

Example 18

Fluorescence excitation and emission spectra: Spectroscopic propertiesof EGFP and its variants were also monitored with their fluorescencespectra, measured in a Fluorescence Spectrophotometer (Hitachi Co. Ltd.)with a 1 cm path length quartz cell at room temperature and at 1 μMconcentration in 10 mM Tris and 1 mM DTT (pH 7.4). Slit widths of 3 nmand 5 nm were used for excitation and emission, respectively. Thequantum yield of EGFP variants with different excitation wavelengths wasobtained with a calculation of equation Eq. (12):

$\begin{matrix}{\phi_{p} = {{\phi_{r}\left( \frac{A_{r}}{A_{p}} \right)}\left( \frac{F_{p}}{F_{r}} \right)\left( \frac{n_{p}^{2}}{n_{r}^{2}} \right)}} & (12)\end{matrix}$

in which, φ_(p) is the relative quantum yield excited at 398 nm or 490nm of EGFP variants; φ_(r) is the relative quantum yield excited at 398nm or 490 nm of the reference sample; A_(p) is the absorption of EGFPvariants at 398 nm or 490 nm; A_(r) is the absorption of the referencesample at 398 nm or 490 nm; F_(p) is the integrated fluorescenceintensity in the range of 500 nm to 600 nm excited at 398 nm or 490 nmof EGFP variants; F_(r) is the integrated fluorescence intensity in therange of 500 nm to 600 nm excited at 398 nm or 490 nm of the referencesample; n_(p) is the refractive index of EGFP variants; and n_(r) is therefractive index of the reference sample.

EGFP was used as the reference sample in the measurement of quantumyield of EGFP variants.

Example 19

Statistical analysis: Statistical analysis was performed with thesoftware package Super ANOVA (Abacus Concepts, Berkeley, Calif.). Valueswere expressed as mean±SEM. Control and treatment groups were comparedby performing an analysis of variance (ANOVA). Fisher's Protected LeastSignificance Difference Test (Fisher's PLSD) was employed for post-hoctests of statistical significance. Significance levels compared to day 1are indicated as follows: *p≦0.05; **p≦0.01; ***p≦0.001.

Example 20

Design of EGFP-based calcium binding proteins: Two different types ofcalcium binding sites were created in enhanced green fluorescent protein(EGFP). FIG. 9A shows the design of EGFP-D2 (SEQ ID No.: 64) containinga discontinuous calcium binding site based on common pentagonalbipyramidal geometry and chemical properties (J. Am. Chem. Soc. 127:2085-2093; J. Am. Chem. Soc. 125: 6165-6171). It was formed by oxygenfrom five negatively charged ligand residues from sidechain carboxylgroups by the mutated amino acid positions, S2D L194E, S86D, and thenatural ligands of D82 and E5. FIG. 9A also shows the engineering of acontinuous calcium binding site EGFP-G1 (SEQ ID No.: 4) by integratingthe EF hand calcium binding motif III of calmodulin inserted on loop 9between residues E172 and D173 of EGFP. In addition to fulfilling therequired criteria for calcium binding to have proper local calciumbinding geometric properties and charge arrangement, these calciumbinding sites were also selected based on criteria to assist chromophoreformation: (i) site location and residue mutations should not abolishthe chromophore synthesis or folding of the protein. Any residues thatare conserved in fluorescent proteins and essential for proteinstructure and folding are not mutated; (ii) the location should be in asolvent-exposed region to have a good accessibility to enable calciumbinding; (iii) to avoid drastic alterations of protein folding andchromophore formation by the introduced charged calcium ligand residues,a calcium binding pocket with few mutations necessary is preferred.

Additional mutants were also created to test the effect of foldingmutations on the fluorescence at both temperatures. The cycle 3mutations were applied in sets of two or three of each calcium bindingsite to examine the differences in fluorescence in accordance with theapplied mutations. Two mutations, M153T and V163A, were applied toEGFP-D2 and EGFP-G1 to create EGFP-D2-C2 and EGFP-G1-C2 (SEQ ID No.: 19)constructs, respectively. The last mutation F99S was furtherincorporated to create the C3 constructs, EGFP-D2-C3 and EGFP-G1-C3 (SEQID No.: 34). The same mutations (C2 and C3) were also applied toEGFP-wt.

FIG. 9B illustrates a model structure of modified grafting EGFP sensor.One EF-hand was inserted in the fluorescent sensitive location of EGFP,generating EGFP-G1. A site-directed mutagenesis on the beta-sheetsurface introducing a negatively charged residue to form a Ca²⁺ bindingsite with three existed negatively charged residues.

Bacteria expression of the EGFP calcium binding proteins: The nineproteins, EGFP, EGFP-C2, EGFP-C3, EGFP-D2 (SEQ ID No 64), EGFP-D2-C2,EGFP-D2-C3, EGFP-G1 (SEQ ID No.: 4), EGFP-G1-C2 (SEQ ID No.: 19), andEGFP-G1-C3 (SEQ ID No.: 34) were first expressed in bacteria at 30° C.and 37° C. to examine the differences in the chromophore maturation bymonitoring the fluorescence intensity at 510 nm (excited at 490 nm).Average intensities of the nine proteins were taken at five time pointsthroughout the expression.

FIG. 10 lists the average fluorescence intensities for 22 hrs after IPTGinduction. The differences between the 30° C. and 37° C. expressionfluorescence intensities were also calculated. As shown in FIG. 10, at30° C. the addition of both types of calcium binding sites into EGFPdoes not alter the chromophore formation. However, the fluorescentintensities of expressed properties in bacteria were significantlydecreased.

The fluorescent intensities of both EGFP-D2 (SEQ ID No.: 64) and EGFP-G1(SEQ ID No.: 4) is significantly lower than in EGFP at both 30° C. and37° C. The C2 and C3 mutations in EGFP-D2 (SEQ ID No.: 64) resulted in37- and 18-fold increases of its fluorescence intensity at 30° C.,respectively. The fluorescence intensity increase (6- and 4-fold) wasalso observed with C2 and C3 mutations in EGFP-G1 (SEQ ID No.: 4) at 30°C. However, the similar fluorescence intensity increase was not observedwith C2 and C3 mutations in EGFP at 30° C. The fluorescent intensitiesat 510 nm of the proteins with the addition of calcium binding sites D2and G1 at 30° C. were greater than that at 37° C., respectively. WhileEGFP does not have any significantly difference in fluorescent intensityfor both C2 and C3 variants, the C2 constructs for D2 and G1surprisingly exhibited an increased fluorescence over the C3 variants.Though it is not as low in fluorescence as the protein variants withnone of the cycle 3 mutations added, this indicates that F99S actuallyinterferes with the folding of the protein variant when applied to theM153T/V163A construct.

Example 21

Mammalian cell expression of EGFP-based calcium binding proteins: Theeffect of the C2 and C3 mutations on the expression of EGFP calciumproteins in mammalian cells was also monitored using fluorescencemicroscopy. FIG. 11 shows the fluorescence microscope imaging of theHeLa cells at two day expression at 30° C. and 37° C. after transfectionof EGFP-G1, EGFP-G1-C2, and EGFP-G1-C3. As shown in FIGS. 11A-11C, aftertwo days transfection and expression at 30° C., EGFP-G1 (SEQ ID No.: 4)variant and its C2 and C3 mutations were expressed and folded in themajority of the HeLa cells as indicated by their strong fluorescencesignals. However, as shown in FIG. 11D, EGFP-G1 (SEQ ID No.: 4) lost itsfluorescence signal at 37° C. indicating that this temperature was notsuitable to the maturation of EGFP-G1 (SEQ ID No.: 4) in HeLa cells. Incontrast, the addition of C2 and C3 mutations in EGFP-G1 (SEQ ID No.: 4)resulted in a maturation of the proteins at 37° C. in HeLa cells, asshown in FIGS. 11E and 11F.

FIGS. 12A and 12B show the quantitative analysis of fluorescenceintensity of HeLa cells (more than 20 cells per image) transfected withboth EGFP-D2 and EGFP-G1 series at both 30° C. and 37° C. A lowfluorescence intensity of HeLa cells transfected with EGFP-D2 (SEQ IDNo.: 64) was observed at both 30° C. and 37° C. (FIG. 12A) compared withthat of EGFP-G1 (SEQ ID No.: 4). The C2 mutation in EGFP-D2 resulted inthe increase of fluorescence intensity, but further increase was notobserved in the C3 mutation. This result with mammalian cellscorresponded with that observed in E coli. A similar result was alsoindicated with a C2 mutation in EGFP-G1 at 37° C. although the effect ofC2 and C3 mutations of EGFP-G1 was not observed at 30° C., shown in FIG.12B.

Example 22

Spectroscopic properties of the calcium binding GFPs: To further explorethis phenomenon, the proteins were purified. EGFP-D2-C2 and EGFP-G1-C2(SEQ ID No.: 19) were much harder to purify than the parent proteins atthe increased concentrations of the protein, indicating that the proteinfolds more efficiently and there were more soluble fractions that couldeasily be released during sonication. This was expected as EGFP-D2-C2and EGFP-G1-C2 (SEQ ID No.: 19) had 37- and 19-fold higher fluorescencethan their counterparts with no “folding mutations”.

Spectroscopic properties of EGFP-based Ca²⁺ binding proteins wereinvestigated using purified proteins. FIGS. 13A and 13B show the visibleabsorbance and fluorescence emission spectra of EGFP, EGFP-D2 (SEQ IDNo.: 64), and EGFP-G1 (SEQ ID No.: 4) at pH 7.4.

TABLE 5 Spectroscopic property of EGFP, EGFP-D2, and EGFP-G1 and theirC2 and C3 mutations Extinction Coefficient (ε), M−1cm−1 Quantum Yield398 nm 488 nm 488 nm EGFP 5126.6 55900 0 EGFP-C2 7184.3 55506 0 EGFP-C36672.1 55840 0 EGFP-G1 9228.1 28463 0 EGFP-G1-C2 14063 26999 0EGFP-G1-C3 9906.1 28401 0 EGFP-D2 1291.5 9323.8 0 EGFP-D2-C2 5490.052989 0 EGFP-D2-C3 5404.6 56416 0

Table 5 summarizes the spectroscopic properties of EGFP, EGFP-D2, andEGFP-G1 and their C2 and C3 mutations. As shown in FIG. 13A, a majorabsorbance peak at 488 nm and a minor absorbance peak at 398 nm appearedin the visible spectra of EGFP, indicating that the anionic state ofchromophore was the main form in EGFP. A fluorescence emission peak at510 nm was observed in EGFP fluorescence spectrum (FIG. 13B). Thesimilar spectroscopic properties including both extinction coefficientsand quantum yield constant at 398 nm and 488 nm (Table 1) indicate thatthere is no effect of C2 and C3 mutations on the visible absorptionspectra in EGFP-C2 and EGFP-C3. The formation of a Ca²⁺-binding site byusing three mutated ligands S2D, L194E and S86D and two natural ligandsD82 and E5 of EGFP (EGFP-D2 (SEQ ID No.: 64)) resulted in a decrease ofvisible absorption at both 398 nm and 488 nm as observed in FIG. 13A.Comparing to EGFP, for example, the extinction coefficient at 488 nm ofEGFP-D2 (SEQ ID No.: 64) was decreased from 55900 M⁻¹cm⁻¹ to 9324M⁻¹cm⁻¹. Concurrently, the fluorescence emission peak at 510 nm wasdecreased in its fluorescence spectrum (FIG. 13B) although the quantumyield of EGFP-D2 (SEQ ID No.: 64) was almost same with that of EGFP.Strikingly, both C2 and C3 mutations in EGFP-D2 (SEQ ID No.: 64)reproduced the major absorbance peak at 488 nm and minor absorbance peakat 398 nm similar to that of EGFP (Table 5). Taken together, while thequantum yield is significantly increased for EGFP variants with bothtypes of calcium binding sites, the relative distribution ofionic-neutral states of the chromophore was not altered by the additionof folding mutations.

Example 23

Computational Design: The design of calcium-binding sites used the GFPc3structure, 1b9c, due to its 30,000-fold greater fluorescence than wildtype GFP with expression at 37° C. The potential calcium binding siteswere computationally constructed with the desired oxygen-calcium-oxygenangle, oxygen-calcium distance, ligand type, and number of ligands. Oneanchor Asp and four additional potential ligands from Asp, Asn, Glu, orthe backbone were utilized. The calcium-oxygen length was in the rangeof 2.0 to 3.0 Å. The oxygen-calcium-oxygen angles ranged ±45° from thetheoretical angles of the ideal pentagonal bipyramid geometry(Biochemistry 44: 8267-73; J. Am. Chem. Soc. 127: 2085-2093; J. Am.Chem. Soc. 125: 6165-6171).

Example 24

Cloning and purification of GFP variants: Site-directed mutagenesis wascarried out by the classical polymerase chain reaction with pfu or turbopfu (Invitrogen) and with EGFP DNA as the initial template and theforward primer sequence 5′-ACGGCGACGCGAACCTCGCCGACC-3′ (SEQ ID No.: 106)and the reverse sequence is 5′-CCTCGTCGTTGTGGCGGATCTTG-3′ (SEQ ID No.:107). The linear DNA was ligated with T4 DNA ligase (Promega), and thecircular DNA was amplified in E. coli (either DH5α or Top10) competentcells. The mutations to engineer 177c3 included the above mutations,with the addition of F99S, M153T, and V163A, known as cycle 3 (C3). TheF99S forward and reverse primers 5′-CGCACCATCTCCTICAAGGACG-3′ (SEQ IDNo.: 108) and 5′-CTCCTGGACGTAGCCTTCCC-3′ (SEQ ID No.: 109),respectively. M153T and V163A were made, together with the forwardprimer 5′-GAACGGCATCAAGGCGAACTTCAA-3′ (SEQ ID No.: 110) and the reverseprimer 5′-TTCTGCTTGTCGGCCGTGATATAGA-3′ (SEQ ID No.: 111). The mutationswere carried out utilizing turbo pfu (Stratagene), following themanufacturer's protocol with annealing temperatures of 61° C. for F99Sand 63° C. for 153/163. The DNA was purified with a Qiagen Miniprep kit,and the circular variant DNA was verified by automated sequencing at theGSU core facility.

The vector pcDNA3.1+ (Invitrogen) was utilized during the mutagenesisand for the expression of the protein in mammalian cells in the cytosol.For expression of the protein in the ER, the pcDNA3.1+ vector wasmodified through PCR to contain the calreticulin signal peptide at theN-terminus of the protein and the KDEL retention sequence at theC-terminus. The N-terminal tag from calreticulin, MLLSVPLLLGLLGLAAAD(SEQ ID No.: 112) directs the expression of the gene to commence in theER. The C-terminal tag, KDEL, is a retention sequence that retains theexpressed protein in the ER and does not allow it to be shuffled to theGolgi. The N-terminus tag was inserted in two rounds of PCR with fourprimers due to its length. The proteins were expressed fused to a 6×histidine tag with a pet28a vector (EMD Biosciences) in LB mediumcontaining 30 μg/mL kanamycin. Protein expression was induced at anOD₆₀₀ of 0.6 with 0.2 mM IPTG, and growth was continued for 3-4 hrbefore harvesting by centrifugation at 9500 g for 20 min. After breakingthe cells with sonication, the proteins were dissolved with 8 M urea.The denatured protein was refolded by 10× dilution into the buffer (10mM Tris, 1 mM DTT, 1% glycerol, pH 7.4) and was centrifuged to removecellular debris. The refolded protein was purified using Sephadex G-75size exclusion FPLC (10 mM Tris, pH 7.3) to greater than 95% purity. Theexpression and purity of the protein were analyzed by SDS-PAGE. Theprotein concentration was estimated using a calculated extinctioncoefficient of 21,890 M⁻¹cm⁻¹ at 280 nm. The histidine tag used forpurification did not have any effect on calcium and terbium binding.

Example 25

Terbium fluorescence: All buffers for the metal binding andconformational analysis studies in this work were pretreated withChelex-100 Resin (Bio-Rad). The terbium binding of the proteins wasmeasured with a PTI fluorimeter following the emission at 545 nm with anexcitation at 280 nm. For terbium titration, the initial proteinconcentration was 3 μM in 20 mM PIPES, 10 mM KCl, 1 mM DTT, 1% glycerol,pH 6.8, for proteins GFP.Ca1-3 and 10 mM Tris, 1 mM DTT, 1% glycerol, pH7.4 for GFP.Ca2″. A 1.0 or 5.0 mM stock terbium containing the sameconcentration of protein was added directly into the protein samples.Blank samples consisted of the buffer with increasing terbium withoutprotein. The data were baseline corrected, and the integrated area ofthe peak at 545 nm was fitted by assuming a 1:1 terbium:protein binding(J. Am. Chem. Soc. 125: 6165-6171). The data were also analyzed usingSpecfit/32 (Talanta, 33, 943). Each binding affinity is an average of 4to 6 titrations. To investigate the metal selectivity, GFP.Ca1 andGFP.Ca2′ (3 μM) with 20 μM terbium were incubated with 0.1 and 1 mMcalcium, 10 mM magnesium, or 100 μM lanthanum in 10 mM Tris, 1 mM DTT,1% glycerol, pH 7.4; and the terbium fluorescence of each sample wasmeasured.

Example 26

Calcium binding dye competition: The protein (30 or 40 μM) andRhodamine-5N (approximately 20 μM, Molecular Probes) (J. Biol. Chem.264: 19449-19457) were incubated in 10 mM Tris, 1 mM DTT, 1% glycerol,pH 7.4. A 100 mM CaCl₂ stock containing the same concentrations of dye.Protein was gradually added into the mixture, and the fluorescence wasmeasured with a 1 cm path length cell and an excitation of 552 nm. Afterthe titration, the dye concentration was verified by absorbance at 552nm with an extinction coefficient of 63,000 M⁻¹cm⁻¹. The data wereanalyzed by globally fitting the spectra from 560 to 650 nm usingSpecfit/32 with the metal-ligand-ligand model (Talanta, 33, 943).

Example 27

Mammalian cell transfection: Untransfected HeLa cells were maintained on100 mm tissue culture dishes in filter-sterilized Dubelcco's ModifiedEagle's Medium (DMEM, Sigma Chemical Co.) with 44 mM NaHCO₃, pH 7.2, andwere supplemented with 10% v/v Fetal Calf Serum (FCS, Hyclone), 100 U/mlpenicillin and 0.1 mg/ml streptomycin (Pen/Strep, Sigma) at 37° C. with5% CO₂ in a humidified incubation chamber. The designed protein DNA wassubcloned into pcDNA3.1+ vector (Invitrogen) for expression in mammaliancells through EcoRI and BamHI digestion, followed by ligation with T4DNA Ligase. The DNA, confirmed by automated sequencing, was transfectedinto previously prepared 90% confluent HeLa (HEK293, Vero, or CHO) cellsusing Lipofectamine 2000 (Invitrogen) on 60 mm cell-culture-treateddishes. The DNA (3 μg) was mixed with Lipofectamine 2000 in a 1:3 ratioin Opti-MEMI serum-free medium (Invitrogen) and was allowed toequilibrate at room temperature for 20 min in the dark before beingadded to the cells in Opti-MEMI medium. The transfection was allowed toproceed for 4 hrs at 37° C. and 5% CO₂. The transfection medium wasremoved and was replaced with DMEM, 10% FCS, 1% Penicillin-StreptomycinSolution; and the cells were grown at 30° C. at 5% CO₂ for 72 hrs.Mock-transfected HeLa cells were treated in the same way without DNAaddition for a background control.

Example 28

Microscopy Imaging: HeLa cells transfected with GFP.Ca1 were imaged 72hrs following transfection. Coverslips with cells were transferred to amicro-incubation chamber (model MSC-TD, Harvard Apparatus, Holliston,Mass.). Briefly, imaging of GFP.Ca1 fluorescence was performed on aNikon TE300 (Nikon Inc., Melville, N.Y.) inverted microscope equippedwith a Nikon filter block optimized for GFP optics (λ_(ex480),λ_(em 510); Chroma Technology Corp, Rockingham, Vt.), a Metaltek filterwheel (Metaltek Instruments, Raleigh, N.C.) to regulate excitation lightexposure times, a 75 watt xenon short arc lamp, a Hamamatsu CCD digitalcamera (Hamamatsu Corporation, Bridgewater, N.J.), and supported on avibration isolation table. MetaFluor software (Universal Imaging Corp.,v 3.5, Downington, Pa.) was utilized for image acquisition. Acquisitiontime was 50 ms with a gain of 1-3, depending upon the transfectionefficiency.

The fluorescence intensity of the transiently transfected GFP.Ca1 orGFP.Ca1c3 was monitored for several minutes to obtain a baseline valuebefore the addition of ionomycin to the bath buffer to a finalconcentration of 2 μM. The designed protein's fluorescence was imageduntil the fluorescence intensity was stable (typically 2 min), and theintracellular calcium concentration was then manipulated by thesubsequent addition of concentrated CaCl₂ to obtain the targetedextracellular calcium concentration. Multiple additions of CaCl₂ weretypically spaced 1 min apart. Extracellular calcium concentrations werereturned to basal levels by bath perfusion of HBSS⁺⁺ buffer. EGFPwithout a calcium binding site was utilized as a control.

To test the calcium response of the sensor expressed in the ER, 50-100μM ATP and 100 μM histamine were added to the bathing medium to inducecalcium release from the ER. Higher concentrations of ionomycin (2.5-5μM) were utilized to permeabilize the ER membrane and to allow forcalcium uptake with addition of calcium to the bathing medium (10-100mM). Thapsigargin (1 μM) and calmidozolium (2 μM) were added to thebathing medium to empty slowly the ER of calcium.

Example 29

Application of engineered variants of EGFP as analyte sensors with highaffinity and selectivity for Pb²⁺ and Gd³⁺ ions: Toxic metals (e.g.Gd³⁺, La³⁺, Tb³⁺, Pb²⁺, Sm³⁺, Sr²⁺, Hg²⁺ and Cd²⁺) can interactadversely with biological systems. While the toxicological effects ofmetals have been extensively studied, the mechanisms of toxicityrelative to interaction with proteins are not fully-understood. Lead(Pb²⁺) is a persistent, anthropogenic toxic metal responsible for avariety of health problems related to neurological disorders, anemia,kidney damage, hypertension and male fertility decrease (Reprod.Toxicol. (2005). 20: 221-228; (2000) Am. J. Ind. Med. 38: 310-315;(2005) Neurotoxicol. Teratol. 27: 245-257; (1997) Annu. Rev. Nutr. 17:37-50; (2001) Int. J. Toxicol. 20: 113-120; (2000) Int. J. Dev.Neurosci. 18: 791-795; (1987) Ann. N. Y. Acad. Sci. 514: 191-203).Lanthanides are known to block calcium channels in human and animalcells, and Pb²⁺, Cd²⁺, and Hg²⁺ will specifically target voltage-gatedcalcium channels ((2003) J. Bioenerg. Biomembr. 2003. 35: 507-532).There is, therefore, a strong need to develop inexpensive, benignmaterials for the detection and neutralization of toxic metals innatural systems, and for biological remediation. The present disclosure,therefore, encompasses the application of the engineered variants ofEGFP of the disclosure as analyte sensors with high affinity andselectivity for such as Pb²⁺ and Gd³⁺ ions. The autofluorescence of GFPand its variants make it a versatile tag for metal-binding studies wherethe close proximity of a metal cation to a chromophore in the proteinresults in a detectable quenching of the fluorescent peaks ((2000)Biochem. Biophys. Res. Commun. 268: 462-465.)

Example 30

Development of EGFP-Based Pb²⁺ and Ln³⁺ Sensors: EGFP protein variantsdesigned for metal-binding and protease studies were developed viasub-cloning using PCR. Proteins were prepared for purification on aNi²⁺-chelating sepharose column by the addition of a 6×His-tag. Thesevariants provide the scaffold for mutagenesis studies to provide proteinvariants with high metal selectivity, and for use as a protease sensor.EMD Omnipur tris(hydroxymethyl)aminoethane (EMD Chemicals, Inc.,Gibbstown, N.J.), or TRIS, was the buffering agent for the expressedproteins.

Transformation: Recombinant pET28a vector comprising regions encodingEGFP variants were transformed into E. coli cell strain DE3 by heatshock for 90 s at 42° C. The sample was placed on ice for 2 minutes. LBMedium (50 μL) was added and the sample incubated for 30 mins at 37° C.before plating on selective media.

Expression: Kanamycin was used at 0.03 mg/mL. 1.0 L LB media cultureswere incubated to an A₆₀₀ of0.6±0.1isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to aconcentration of 0.2 mM, and the temperature reduced to 20-25° C. 1.0 mLsamples were removed every hour for three hrs, followed by a finalsample on the following day, to evaluate protein expression usingSDS-PAGE gels. Cells were harvested and stored at 4° C.

Purification: A cell pellet was suspended in about 20 mL of extractionbuffer (20 mM TRIS, 100 mM NaCl, 0.1% Triton x-100) and placed on ice.The sample was sonicated 6×30 s periods, with about 5 min intervalsbetween sonications and centrifuged for 20 min at about 5×10⁴ g. Thesupernatant was filtered with 0.45 μm pore size filter (Whatman, FlorhamPark, N.J.) and diluted with the appropriate binding buffer prior toinjection into an FPLC system.

Purification of EGFP variants was completed using an Aktaprime FPLC(Amersham Biosciences, Piscataway, N.J.) equipped with a UV detector anda 280 nm optical filter. For most purifications, a Hitrap 5 mL HPChelating sepharose column was used. The binding Buffer A was 1 MK₂HPO₄, 1 M KH₂PO₄, 250 mM NaCl, pH 7.4 and elution Buffer B was ofBuffer A and 0.5 M imidazole.

The column was first rinsed with 100 mM EDTA, 1 M NaCl, pH 8.0 to removemetals, and rinsed with distilled water. The column was then washed with0.1 M NiSO₄ to bind Ni²⁺ onto the column, which was rinsed again withdistilled water to remove unbound NiSO₄.

For additional protein purification, a Hitrap Q Ion Exchange column (GEHealthcare, Piscataway, N.J.) was used. The binding Buffer A was 20 mMTRIS, pH 8.0 and the elution Buffer B was of 20 mM TRIS, 1 M NaCl, andpH 8.0.

Protein injections onto the column were limited to 5-8 mL and elutedbound protein was collected in 8 mL fractions that were then furtherpurified by dialysis in 2.0 L of 10 mM TRIS, 1 mM Dithiothreitol, pH7.4. Protein fractions were dialysed in dialysis bags with a molecularweigh cutoff value of 3,500 Da for 72 hrs to remove imidazole and otherimpurities. Purity was evaluated using SDS-PAGE gels.

Example 31

Spectroscopic Analysis: Fluorometric spectral analyses of EGFP variantswere conducted with excitation slit widths set at 1 nm, to reducephotobleaching of the proteins, and the emission slit widths were set at2 nm. Excitation wavelengths of 398 nm and 490 nm were used. Data fromthe fluorometers were collected at 1 nm intervals.

The selectivity of the EGFP-based sensors for Pb²⁺ and Gd³⁺ was examinedby monitoring the change of the fluorescence ratioF_((398nm))/F_((490nm)) obtained with 1.0 mM Ca²⁺ in the presence of thetest metal ions.

The ratiometric change from the metal-free protein to the metal-proteincomplex was calculated by integrating the peak areas for each of theemissions scans (398 nm and 488 nm) from 500-600 nm as a sum of theintensities recorded at each 1 nm interval, and then evaluating theratio of (F398/F488), as seen in Eq. 13.

$\begin{matrix}\begin{matrix}{{{Ratiometric}\mspace{14mu} {change}} = \left( {F\; {398/F}\; 488} \right)} \\{= \left( \frac{\sum\limits_{500}^{600}{{Counts}\; 398}}{\sum\limits_{500}^{600}{{Counts}\; 488}} \right)}\end{matrix} & \left( {{Eq}.\mspace{14mu} 13} \right)\end{matrix}$

The ratio eliminates possible errors associated of absolute intensityvalues due to instrumental variations.

The fluorescent ratiometric change (F398/F490) was evaluated for 1.0 μMof the EGFP variants to evaluate selectivity between Ca²⁺ and eitherPb²⁺ or Gd³⁺ in 10 mM TRIS-Cl, pH 7.4. First, 1 mM Ca²⁺ was added to theprotein, followed by aliquots of the competing metal.

The affinity of a competing ion was assumed to be directly proportionalto the change in the ratio (F398/F490), as calculated using Equation 11.To calculate the K_(eq) for the competitive titration, the value for thefraction of the competing ion (F) was normalized across the range ofconcentrations evaluated. This F Normalized value (F_(Norm)) wascalculated with Eq. 14.

F _(Norm)=(F _(Ca initial) −F _(M+))/(F _(Ca initial) −F_(M+final))  (Eq. 14)

In Equation 3, F_(Ca initial) is the initial ratio (F398/F490) followingaddition of Ca²⁺, F_(M+) is the ratio at each point of addition of acompeting metal ion, and F_(M+final) is the ratio at the finalconcentration of metal. K for the competitive titration was thecalculated in using the curve-fitting equation:

F _(Norm)=((([P] _(t) +[M] _(t) +K)−(([P] _(t) +[M] _(t) +K)²−4[P] _(t)[M] _(t))^(1/2))/2[P] _(t))+(C*[M] _(t))  (Eq. 15)

where the final term, (C*[M]_(t)), accounts for non-specific binding.The K_(d) for Pb²⁺ or Gd³⁺ was calculated using Eq. 4, with K calculatedfrom Eq. 15, and the K_(d) for Ca²⁺ which was previously determined forthe EGFP-C2 variant to be 440 μM.

K _(dM+) =K/(1+([Ca²⁺ ]/K _(dCa)))  (Eq. 16)

The absorbance scan encompassed the range of 600-220 nm.

Example 32

It was determined from competitive titrations that Gd³⁺ and Pb²⁺displace Ca²⁺ in the binding sites of the engineered EGFP variants ofthe disclosure. FIG. 18 shows the normalized ratiometric changesassociated with displacement of Ca²⁺ by Pb²⁺. FIGS. 19A to D showchanges in fluorescence intensity resulting from displacement of Ca²⁺ byPb²⁺, and a 2-3 nm red shift in the spectra near 10 μM Pb²⁺. This redshift is believed to be the result of conformational changes relative tothe chromophore, unrelated to displacement of Ca²⁺ which had alreadyoccurred. These data were then used to calculate binding affinities forboth Gd³⁺ and Pb²⁺.

For the EGFP-C2 variant analyzed in this work, the binding affinitiesfor Pb²⁺ and Gd³⁺ were both found to be approximately 200 times higherthan for Ca²⁺ (FIGS. 19B and 19D). For the other EGFP variant (SEQ IDNos.: 18 and 19), the binding affinity for Pb²⁺ was found to be over 100times higher than Ca²⁺. (FIG. 19C). These higher affinities, coupledwith the conformational changes associated with the binding of thesemetals, suggests an important relationship to toxicity. It also providesfor a sensor capable of high-affinity binding for Pb²⁺ and Gd³⁺.

Example 33

CatchER biosensor family design strategy: Based on key determinants forfine-tuning Ca²⁺ binding affinity and Ca²⁺-induced conformationalchanges and the established chromophore properties of fluorescentproteins, Ca²⁺ sensors with fast fluorescence response were designed bycoupling Ca²⁺ binding sites directly to the chromophore rather thanrelying on stretched protein-protein interaction to modulate chromophoreconformation.

The computationally-assisted design was based on the following criteriaand considerations: (i) it requires four or five oxygen ligand atomsfrom protein residues (typically, carboxyl groups of D, E, N, Q)situated in the spherical geometry characteristic of natural Ca²⁺binding proteins; (ii) appropriate choice of residue charge and type canbe chosen to fine-tune Ca²⁺ binding affinity and metal selectivity;(iii) diffusion-limited access of Ca²⁺ to the site requires good solventaccessibility; (iv) propagating Ca²⁺-induced, local conformational andelectrostatic changes to the chromophore can be achieved by properlylocating of the charged ligand residues with respect to it; (v) thesechanges must occur rapidly than the rate of conversion from a neutral toanionic state ascribed to these chromophores; and (v), the createdbinding site must not interfere with the chromophore's synthesis andformation. The EGFP variant with the M153T/V163A mutation (EGFP Cycle 2)was chosen as the scaffold protein because of its high fluorescenceintensity, folding efficiency, and thermostability.

Example 34

Plasmid construction, protein expression, and purification: Bacterialexpression plasmids for EGFP variants D8 to D12 were constructed bysite-directed mutagenesis on cycle 2 EGFP (F64L/S65T/M153T/T163A)inserted in the pET28a vector (EMD Biosciences, San Diego, Calif.)vector between the BamHI and EcoRI restriction enzyme cleavage sites.The DNA sequence of the designed EGFP variants between these tworestriction sites were cleaved and inserted into pcDNA3.1+ vector(Invitrogen, Carlsbad, Calif.). Calreticulin ER targeting sequence(CRsig) MLLSVPLLLGLLGLAAAD (SEQ ID No.: 112) and ER retention sequenceKDEL were added to the N- and C-termini, respectively, to construct themammalian cell expression plasmids. CatchER (D11) and its variants(D8-D10 and D12) were bacterially expressed in Escherichia coliBL21(DE3) and purified using established methods (Heim & Tsien (1996)Curr. Biol. 6:178-182; Zou et al., (2007) Biochemistry 46: 12275-12288).

Example 35

In situ measurement of CatchER's Ca²⁺ dissociation constant: CatchER'sCa²⁺ dissociation constant (K_(d)) was measured in BHK and C2C12 cells.ER Ca²⁺ in BHK cells was depleted by applying 100 μM histamine and 5 μMthapsigargin in Ringer 0 Ca²⁺ buffer. Cells were permeabilized in 100 μMdigitonin in intracellular-like solution containing 140 mM KCl, 10 mMNaCl, 1 mM MgCl₂, 20 mM Hepes, pH 7.25. Calibration buffers wereprepared by adding Ca²⁺ to the intracellular-like solution, reachingfinal concentrations of 0.05, 0.1, 0.5, 1, 5, and 10 mM, and 200 μM EGTAbuffer. F_(min) and F_(max) were determined in 200 μM EGTA and 10 mMCa²⁺ with no Ca²⁺ ionophore, respectively.

Similar in situ K_(d) calibration was conducted in C2C12 myoblasts. ERCa²⁺ of permeabilized cells was depleted in intracellular buffercontaining 10 μM IP3 and 2 μM thapsigargin. For calibration, 1, 3, 10,and 20 mM Ca²⁺ buffers were applied in the presence of 5 μM ionomycin.F_(min) and F_(max) were determined in 3 mM EGTA and 20 mM Ca²⁺,respectively.

The fluorescence was normalized according to the equation:

$f = \frac{F - F_{\min}}{F_{\max} - F_{\min}}$

and K_(d) determined by the Hill-equation:

$f = \frac{\left\lbrack {Ca}^{2 +} \right\rbrack^{n}}{K_{d} + \left\lbrack {Ca}^{2 +} \right\rbrack^{n}}$

The Kd was 1.07±0.26 mM (0.90±0.19 Hill coefficient) in BHK cells and1.09±0.20 mM (0.94±0.17 Hill coefficient) in C2C12 cells.

Example 36

Kinetic analysis of Ca²⁺ binding to CatchER by stopped-flow: Thefluorescence kinetics of bacterially expressed CatchER was investigatedusing an SF-61 stopped-flow spectrofluorometer (Hi-Tech Scientific,Salisbury, UK; 10-mm path length, 2.2-ms deadtime at room temperature)at 22° C. Fluorescence intensity changes were recorded with a 455 nmlong-pass filter with excitation at 395 nm. Equal volumes of Ca²⁺-freeprotein in 10 mM Tris-Cl at pH 7.4 and Ca²⁺ in the same buffer weremixed in the stopped-flow spectroflurometer, yielding finalconcentrations of 10 μM CatchER and 50, 100, 200, 300, 500, and 1000 μMCa²⁺. The stopped-flow traces were fit to Eq. (1), which describes F,the fluorescence intensity at any given time; F_(∞), the fluorescence atinfinite time; and ΔF, the amplitude of the fluorescence change.

F=F _(∞) −ΔFexp(−k _(obs) ·t)  17)

F=F _(∞) +ΔFexp(−k _(obs) ·t)  18)

k _(obs)·τ=ln 2  19)

Example 37

Apparent pK_(a) determination by pH profile: The apparent pK_(a) ofCa²⁺-free or Ca²⁺-loaded CatchER was determined with bacteriallyexpressed protein by fitting the fluorescence intensity change at 510 nm(λex=488/395 nm). 5 μM protein was dissolved in different buffers withpH ranging from 4.5 to 9.5 in the presence of either 10 μM EGTA (apo) or4 mM Ca²⁺ (holo), and the actual pH was determined after measuringfluorescence. The proposed interaction scheme is

$\begin{matrix}{{pH} = {{pKa} + {\log \frac{\lbrack P\rbrack}{\left\lbrack {HP}^{+} \right\rbrack}}}} & \left. 20 \right) \\{f = \frac{F - F_{\min}}{F_{\max} - F_{\min}}} & \left. 21 \right) \\{F_{\min} = {\lbrack P\rbrack_{T}c_{1}}} & \left. 22 \right) \\{F_{\max} = {\lbrack P\rbrack_{T}c_{2}}} & \left. 23 \right) \\{F = {{\left( {\lbrack P\rbrack_{T} - \lbrack P\rbrack} \right)c_{1}} + {\lbrack P\rbrack c_{2}}}} & \left. 24 \right) \\{f = {\frac{{\lbrack P\rbrack_{T}c_{1}} - {\lbrack P\rbrack c_{1}} + {\lbrack P\rbrack c_{2}} - {\lbrack P\rbrack_{T}c_{1}}}{{\lbrack P\rbrack_{T}c_{2}} - {\lbrack P\rbrack_{T}c_{1}}} = \frac{\lbrack P\rbrack}{\lbrack P\rbrack_{T}}}} & \left. 25 \right) \\{\frac{\lbrack P\rbrack}{\left\lbrack {HP}^{+} \right\rbrack} = \frac{1}{{1/f} - 1}} & \left. 26 \right) \\{f = \frac{1}{1 + {\exp\left( \frac{{pKa} - {pH}}{c} \right)}}} & \left. 27 \right)\end{matrix}$

H⁺ is the proton; P is the CatchER protein; f, the normalized ΔF change;[P]_(T), the total protein concentration; c₁ or c₂ is the extinctioncoefficient of HP⁺ or P fluorescence, respectively; F is the real-timefluorescence; F_(min), the fluorescence at the lowest pH; F_(max), thefluorescence at the highest pH; c is a constant for adjustment. Thevalue theoretically equals Ige. The apparent pK_(a), fitted by a singleexponential (Eq. 11), were 7.59±0.03 and 6.91±0.03 for apo and holoforms excited at 488 nm and 7.14±0.02 and 6.95±0.06 at 395 nm,respectively.

Example 38

CatchER:Ca²⁺ stoichiometry studied by the Job Plot: The stoichiometry ofthe CatchER and Ca²⁺ interaction was determined at the maximal relativeamount of Ca²⁺-bound CatchER in the Job Plot (15). Ca²⁺-free and bound[CatchER] were converted to fluorescence intensity following theequation: F=S_(f)·C_(f)+S_(b)·C_(b), where F is the apparentfluorescence intensity; S_(f) and S_(b) are the coefficients of Ca²⁺free and bound CatchER, respectively; and C_(f) and C_(b) are theconcentration of Ca²⁺ free and bound CatchER, respectively. The relativeamount of Ca²⁺ bound CatchER (C_(b)·V, V=1) was calculated using the Eq.(12). Fluorescence emission (λ_(ex)=488/395 nm) and absorbance spectrawere recorded with [CatchER]: 28.7, 23.3, 19.4, 15.1, 11.6 μM inresponse to [Ca²⁺]: 11.3, 16.7, 20.6, 24.9, 28.4 μM, respectively.

$\begin{matrix}\begin{matrix}{\frac{F_{{Ca}^{2 +} - {bound}}}{F_{{Ca}^{2 +} - {free}}} = \frac{{S_{f} \cdot C_{f}} + {S_{b} \cdot C_{b}}}{S_{f} \cdot C_{T}}} \\{= \frac{{S_{f}\left( {C_{T} - C_{b}} \right)} + {S_{b} \cdot C_{b}}}{S_{f} \cdot C_{T}}} \\{= {1 + \frac{C_{b} \cdot \left( {S_{b} - S_{f}} \right)}{S_{f} \cdot C_{T}}}}\end{matrix} & \left. 28 \right) \\{a = \frac{S_{b} - S_{f}}{S_{f}}} & \left. 29 \right) \\{{\frac{C_{b}}{C_{T}} \cdot a} = {\frac{F_{{Ca}^{2 +} - {bound}}}{F_{{Ca}^{2 +} - {free}}} - 1}} & \left. 30 \right) \\{{C_{b} \cdot V} = {\left( {\frac{F_{{Ca}^{2 +} - {bound}}}{F_{{Ca}^{2 +} - {free}}} - 1} \right) \cdot \frac{C_{T}}{a}}} & \left. 31 \right)\end{matrix}$

Example 39

NMR Spectroscopy: All NMR experiments were performed at 37° C. using aVarian 800 or 600 MHz spectrometer. Typically, NMR samples contained 0.3mM ¹⁵N- or ¹³C, ¹⁵N-labeled protein in 10 mM Tris, 10 mM KCl, 10% D₂O,pH 7.4. For backbone assignment of ¹H, ¹³C, and ¹⁵N resonances, a HNCAwas collected on a Varian (nova 800 MHz spectrometer, and a CBCA(CO)NHwas collected on a Varian (nova 600 MHz spectrometer, both equipped witha cryogenic probe. For Ca²⁺ titration, {¹H, ¹⁵N} HSQC spectra werecollected, and chemical shift perturbations calculated using theequation Δδ_(av)={0.5[Δδ(¹H^(N))²+(0.2Δδ(¹⁵N))²]}^(1/2), where Δδ is thechange in chemical shift between the apo and Ca²⁺-loaded form.Rotational correlation time (τ_(c)) was measured using a shared,constant-time, cross-correlated relaxation (SCT-CCR) pulse sequence. Inthis measurement, a series of highly sensitive HSQC spectra werecollected at relaxational acquisition times from 0 to about 100 ms.Residue-specific τ_(c) values were then extracted from the exponentialdecay rates. T1 and T2 were collected on a Varian (nova 600 MHzspectrometer. Integrations of peak collected at 0, 30, 60, 100, 240,480, 720, 1000, and 1500 ms (T1) and 10, 30, 50, 70, 90, 110, 130, and150 ms (T2) were fitted with I=I₀exp(−t/T_(1/2)), where I₀ is theintensity at zero decay, and t, the relaxation decay. τ_(c) values werecalculated following the equation below:

τ_(c)=(2ω_(N))⁻¹·√{square root over ((6T ₁ /T ₂−7))}  32)

ω_(N)=2π·f _(N)  33)

Example 40 Structure Analysis of Catcher and its Variants by NMR andVerified by X-Ray

The reality of calcium binding to the sensor can be further proved bycalcium titration by NMR after the construction of the relationshipbetween fluorescence intensity and calcium concentration. The conditionof sample preparation is very important to affect the quality of the NMRspectra as the protein is the beta-sheet protein which has the tendencyto be aggregation. One of the factors is the temperature that willinfluence the desperation of the peaks, so we test the D11 spectraquality in different temperatures in 500 MHz NMR. FIG. 32 shows thetemperature dependent NMR HSQC spectra changes of CatchER.

The peak number increases from 128 to 194 along with the temperatureraise from 20° C. to 37° C. As the total amino acid number of EGFP is238, the optimal temperature for the experiment operation is above 37°C.

Prior to the gNhsqc calcium titration, 1D NMR calcium titration wasoperated to roughly detect chemical shifts and besides the sidechainsdispersed around 0 to 6 ppm, the major chemical shifts of the NH groupwere approximate 6.6 to 7.8 ppm which was later proved to be fromsidechain NH. The region of 8 to 11 ppm did not exhibit obvious shiftdue to the huge number of peaks overlap together what was insensitive tobe distinguished.

gNhsqc is conducted to verify the chemical shift of each residues as 1Dexperiment is not effective enough to explore such a huge protein. Theprotein with the concentration of 0.3 mM is dissolved in 10 mM pH 7.4Tris buffer and 10% of D₂O for the final concentration. The operationtemperature is 40° C. for 600 MHz NMR. FIG. 33 shows a 1D NMR spectra ofchemical shift changes of CatchER triggered by Ca²⁺.

Salt effect should firstly be examined to verify whether D11 cannonspecifically bind to monovalent cations. 0.1 mM EGTA was added intothe sample as the starting points and then titrated with 10 mM KCl tomonitor the chemical shifts. After these two spectra overlapped, theyperfectly matched. It hinted that high concentration salt could notcause conformation change of the protein so that there is no nonspecificbinding.

X-ray crystal structure of Ca2+_free_CatchER, Ca2+_loaded CatchERCa2+-free CatchER exhibited a major absorption peak at 395 nm and aminor peak at 490 nm, which is similar to the wtGFP, with a ratio of 395nm to 490 nm 3.0 measured in vitro (Tang, et. al.). From the crystalstructures of Ca2+ free and loaded form CatchER (FIG. 34), the sidechainof 222 rotated to change the distance of hydrogen bonds between carboxylgroup of sidechain Glu222 and Ser205 and hydroxyl group of chromophore.The proposed hydrogen networks surrounding the chromophore are based onthe previous reported crystal structure of wtGFP (pdb code: 1EMB) andEGFP (pdb code: 1EMA). The previous reported wtGFP from A jellyfish hastwo absorption peaks at 390 nm (major) and 490 nm (minor), suggesting amixture of two forms of chromophore co-existed in one fluorescentprotein (Heim, 1996). Though from the DNA sequence alignment, the sitedirected mutagenesis S65T is the cause of the major difference, however,the chromophore of wtGFP and EGFP can be overlapped well from thecrystal structure, but the sidechain surrounding the chromophoreexhibited different conformation, especially for Thr203 and Glu222(Baird, 1997). In wtGFP, the mainchain of Thr203 distantly interactswith the chromophore through a water molecule (Remington, 1996 andTsien, 1998), while for EGFP, the polar sidechain hydroxyl oxygeninteract oxygen atom of chromophore directly, forming a short hydrogenbond 2.5 A, and stabilize the chromophore at anionic form, causing amajor absorption peak at 490 nm. This stabilization is further enhancedby the special orientation of sidechain carboxyl group of E222, the onlynegative charged residue protrude toward the chromophore, forming arestricted hydrogen network among E222, V61 and T65 conjugated withinthe chromophore. However, the oxygen of Tyr66 of wtGFP only directlyinteracts with H2O without forming hydrogen bonds with polar residues,maintained neutral form, contributing to the major absorption peak at395 nm. The two oxygen atoms of the carboxyl group of E222 are equallypartially charged forming hydrogen bonds with Ser205 and chromophore. Ahydrogen bridge between the hydroxyl group of T65 and Y66 is formed viaE222, S205 and a water molecule, ensuring an efficient electron transferbetween polar residues within the chromophore. An interestingobservation of carboxyl group of E222 rotating between Ca2+ free andloaded form CatchER from the crystal structure, altering the distance ofthe hydrogen bonds between the carboxyl group of E222 to S205 andchromophore. Up to now, the rotation of Glu222 triggered by analytebinding has not been reported, especially correlated with the opticalproperties change. It is possible that the E222 sidechain rotation inresponse to Ca2+ may contribute to the fast kinetics of CatchER as asingle residue rotation is faster than long-range protein-proteininteraction in FRET pair based sensor. However, this sidechain rotationof Glu222 was not observed in comparison of Gd3+_free and Gd3+_soakingCatchER structure (FIG. 35, it is plausible that the residues buriedinside the beta-barrel structure of GFP did not exhibit furtherconformational change after crystallization, even during the metalsoaking, though fluorescent intensity of CatchER was dramaticallyenhanced after adding Gd3+ during in vitro titration. The other keyresidue Thr203 of CatchER maintained one hydrogen bond between the mainchain oxygen and water close to chromophore in all of metal-free,Ca2+_loaded and Gd3+_loaded form CatchER, similar to the wtGFP,suggesting that the fluorescent intensity of Ca2+_loaded CatchER onlyrecover 50% of EGFP is possibly due to the fixe hydrogen bond networkclose to the phenol group of Tyr66 of chromophore maintained similar towtGFP.

<SEQ ID No.: 1; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 2; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 3;PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 4; PRT3;Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 5; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu<SEQ ID No.: 6; PRT3; Artificial sequence>MetSerValLeuThrProLeuLeuLeuArgGlyLeuThrGlySerAlaArgArgLeuProValProArgAlaLysIleHisSerLeuGlySerGlyProSerArgMetValSerLysGlyMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyr8GlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeu160ValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsn1IleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisVal240MetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGln2AsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys310 <SEQ ID No.: 7; PRT3;Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluSerGluGluGluLysArgGluAlaGluArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisAlaAlaThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 8; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAsnLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 9; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAsnLysAsnGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 10; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 11; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAspGlyThrIleThrThrLysGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 12; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerCysAspLysPheLeuAspAspAspIleThrAspAspIleMetCysAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 13; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerAlaAspLysPheLeuAspAspAspIleThrAspAspIleMetCysAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 14; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerCysAspLysPheLeuAspAspAspIleThrAspAspIleMetAlaAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 15; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerAlaAspLysPheLeuAspAspAspIleThrAspAspIleMetAlaAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 16; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 17; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAsp24HisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 18;PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleVa124LeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 19; PRT3;Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 20; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsn16IleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGln24AsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu<SEQ ID No.: 21; PRT3; Artificial sequence>MetSerValLeuThrProLeuLeuLeuArgGlyLeuThrGlySerAlaArgArgLeuProValProArgAlaLysIleHisSerLeuGlySerGlyProSerArgMetValSerLysGlyMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeu16ValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisVal24MetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys31 <SEQ ID No.: 22; PRT3; Artificialsequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerGluGluGluLysArgGluAlaGluArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisAlaAlaThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 23; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAsnLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 24; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAsnLysAsnGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 25; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnl6GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 26; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAspGlyThrIleThrThrLysGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 27; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerCysAspLysPheLeuAspAspAspIleThrAspAspIleMetCysAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 28; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerAlaAspLysPheLeuAspAspAspIleThrAspAspIleMetCysAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 29; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerCysAspLysPheLeuAspAspAspIleThrAspAspIleMetAlaAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 30; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerAlaAspLysPheLeuAspAspAspIleThrAspAspIleMetAlaAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 31; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 32; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAsp24HisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 33;PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleVal24LeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 34; PRT3;Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlysTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 35; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsn16IleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGln24AsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu<SEQ ID No.: 36; PRT3; Artificial sequence>MetSerValLeuThrProLeuLeuLeuArgGlyLeuThrGlySerAlaArgArgLeuProValProArgAlaLysIleHisSerLeuGlySerGlyProSerArgMetValSerLysGlyMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeu16ValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisVal24MetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys31 <SEQ ID No.: 37; PRT3; Artificialsequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerGluGluGluLysArgGluAlaGluArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisAlaAlaThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 38; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAsnLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 39; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAsnLysAsnGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 40; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 41; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspLysAspGlyAspGlyThrIleThrThrLysGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 42; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAsppheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerCysAspLysPheLeuAspAspAspIleThrAspAspIleMetCysAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 43; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerAlaAspLysPheLeuAspAspAspIleThrAspAspIleMetCysAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 44; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerCysAspLysPheLeuAspAspAspIleThrAspAspIleMetAlaAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 45; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluSerArgAsnIleCysAspIleSerAlaAspLysPheLeuAspAspAspIleThrAspAspIleMetAlaAlaLysLysIleLeuAspIleLysGlyAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeu24SerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 46; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnAspLys16AspGlyAsnGlyTyrIleSerAlaAlaGluLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 47; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnGluGlu16GluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 48; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnAspLys16AspGlyAsnGlyTyrIleSerAlaAlaGluLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 49; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnGluGlu16GluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 50; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnAspLys16AspGlyAsnGlyTyrIleSerAlaAlaGluLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAla24GlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 51; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnGluGlu16GluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 52; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 53; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnThrGluGluGlnIleAlaGluPheLysGluAlaPheSerLeuPheAspLysAspGlyAspGlyThrIleThrThrLysGluLeuGlyThrValMetArgSerIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSer24AlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 54; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 55; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnThrGluGluGlnIleAlaGluPheLysGluAlaPheSerLeuPheAspLysAspGlyAspGlyThrIleThrThrLysGluLeuGlyThrValMetArgSerIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSer24AlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 56; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer24LysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys27<SEQ ID No.: 57; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnThrGluGluGlnIleAlaGluPheLysGluAlaPheSerLeuPheAspLysAspGlyAspGlyThrIleThrThrLysGluLeuGlyThrValMetArgSerIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSer24AlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 58; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValAspPheGluGlyAspThrLeuAsnAsnAspIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 59; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleAsnValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuAsnAsnAspIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 60; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnAspGluAspGlyAspValAsnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 61; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetAspGlnHisAspPhePheAspSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 62; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetAspGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 63; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisAsnLysGlnAspAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnAspTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyAspThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 64; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 65; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsn16IleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArg24AspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu<SEQ ID No.: 66; PRT3; Artificial sequence>MetSerValLeuThrProLeuLeuLeuArgGlyLeuThrGlySerAlaArgArgLeuProValProArgAlaLysIleHisSerLeuGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGlu16LeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGln24SerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 67; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleAspValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuSerAsnGluIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 68; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspAspPheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrGluMetAspAspLysGlnLysAsn16GlyAspLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 69; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProAspLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePheGluLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnSerLeuGlyHisLysAsnGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGluLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 70; PRT3; Artificial sequence>MetValSerLysGlyGluGluAspPheThrGlyValAsnProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaSerProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysAspGluGlyAspThrAspValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 71; PRT3; Artificial sequence>MetValSerLysGlyGluGluAspPheThrGlyValAsnProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaThrProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysAspGluGlyAspThrGluValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsn16GlyIleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 72; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValAspPheGluGlyAspThrLeuAsnAsnAspIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 73; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleAsnValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuAsnAsnAspIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 74; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnAspGluAspGlyAspValAsnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 75; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetAspGlnHisAspPhePheAspSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 76; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetAspGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 77; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisAsnLysGlnAspAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnAspTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyAspThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 78; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 79; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsn16IleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArg24AspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu<SEQ ID No.: 80; PRT3; Artificial sequence>MetSerValLeuThrProLeuLeuLeuArgGlyLeuThrGlySerAlaArgArgLeuProValProArgAlaLysIleHisSerLeuGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGlu16LeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGln24SerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 81; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleAspValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuSerAsnGluIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 82; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspAspPheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrGluThrAspAspLysGlnLysAsn16GlyAspLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 83; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProAspLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePheGluLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnSerLeuGlyHisLysAsnGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGluLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 84; PRT3; Artificial sequence>MetValSerLysGlyGluGluAspPheThrGlyValAsnProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaSerProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysAspGluGlyAspThrAspValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 85; PRT3; Artificial sequence>MetValSerLysGlyGluGluAspPheThrGlyValAsnProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaThrProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysAspGluGlyAspThrGluValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 86; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValAspPheGluGlyAspThrLeuAsnAsnAspIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 87; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleAsnValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuAsnAsnAspIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 88; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnAspGluAspGlyAspValAsnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 89; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetAspGlnHisAspPhePheAspSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 90; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetAspGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValAsnLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 91; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisAsnLysGlnAspAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnAspTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyAspThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 92; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 93; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsn16IleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArg24AspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu<SEQ ID No.: 94; PRT3; Artificial sequence>MetSerValLeuThrProLeuLeuLeuArgGlyLeuThrGlySerAlaArgArgLeuProValProArgAlaLysIleHisSerLeuGlySerGlyProSerArgMetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysAspAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGlu16LeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGln24SerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.: 95; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleAspValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuSerAsnGluIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnl1GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 96; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspAspPheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrGluThrAspAspLysGlnLysAsn16GlyAspLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValGluLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 97; PRT3; Artificial sequence>MetValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProAspLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIleSerGluLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnSerLeuGlyHisLysAsnGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGluLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 98; PRT3; Artificial sequence>MetValSerLysGlyGluGluAspPheThrGlyValAsnProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaSerProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysAspGluGlyAspThrAspValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 99; PRT3; Artificial sequence>MetValSerLysGlyGluGluAspPheThrGlyValAsnProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaThrProGluGlyTyrValGlnGluArgThrIleSerPheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysAspGluGlyAspThrGluValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsn16GlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQID No.: 100; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspGlySerGlyProSerArg<SEQ ID No.: 101; PRT3; Artificial sequence> LysAspGluLeu <SEQ ID No.:102; PRT3; Artificial sequence>MetSerValLeuThrProLeuLeuLeuArgGlyLeuThrGlySerAlaArgArgLeuProValProArgAlaLysIleHisSerLeuGlySerGlyProSerArg <SEQ ID No.: 103; PRT3; Artificial sequence>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:104; PRT3; Artificial sequence>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:105; PRT3; Artificial sequence>MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu <SEQ ID No.: 106; DNA;Artificial sequence> ACGGCGACGCGAACCTCGCCGACC <SEQ ID No.: 107; DNA;Artificial sequence> CCTCGTCGTTGTGGCGGATCTTG <SEQ ID No.: 108; DNA;Artificial sequence> CGCACCATCTCCTTCAAGGACG <SEQ ID No.: 109; DNA;Artificial sequence> CTCCTGGACGTAGCCTTCCC <SEQ ID No.: 110; DNA;Artificial sequence> GAACGGCATCAAGGCGAACTTCAA <SEQ ID No.: 111; DNA;Artificial sequence> TTCTGCTTGTCGGCCGTGATATAGA <SEQ ID No.: 112; PRT;Artificial sequence> MLLSVPLLLGLLGLAAAD <SEQ ID No.: 113; PRT;Artificial sequence> DKDGNGYISAAE <SEQ ID No.: 114; PRT; Artificialsequence> EEEIREAFRVFDKDGNGYISAAELRHVMTNL <SEQ ID No.: 115; DNA;Artificial sequence>TATTACGTGTTCGCTGGCTaGCGTTTaACTTaAGCTTATGGGGGCCAGAGCAGTGTCCGAGCTGCGGCTGGCACTGCTGTTTGTACTGGTGCTAGGGACGCCCAGGTTAGGGGTCCAGGGGGAAGATGGGTTGGACTTCCCTGAGTACGACGGTGTGGACCGTGTGATCAATGTGAATGCCAAGAACTACAAGAACGTGTTTAAGAAGTATGAGGTGCTGGCCCTCCTCTACCATGAGCCCCCTGAGGACGACAAGGCCTCGCAGAGACAATTTGAGATGGAGGAGCTAATCCTGGAGTTAGCAGCCCAAGTCTTAGAAGACAAGGGTGTTGGCTTTGGCCTGGTGGACTCAGAGAAGGATGCAGCTGTGGCCAAGAAACTAGGACTAACTGAAGAAGACAGCGTTTATGTGTTCAAAGGAGATGAAGTCATTGAATATGACGGCGAGTTTTCTGCAGACACTCTGGTGGAGTTTCTGCTTGATGTCCTAGAAGACCCTGTAGAGTTGATTGAAGGTGAACGAGAGCTGCAGGCATTTGAGAATATTGAAGATGAAATCAAACTCATTGGCTACTTCAAGAGCAAAGACTCAGAGCATTACAAAGCCTACGAGGACGCAGCTGAAGAGTTCCATCCCTACATCCCTTTCTTCGCTACCTTCGACAGCAAGGTGGCAAAGAAGCTGACTCTGAAGTTGAATGAGATTGATTTCTACGAGGCCTTCATGGAAGAGCCTATGACCATCCCAGACAAGCCCAACAGTGAAGAGGAGATTGTGAGCTTCGTGGAGGAGCACAGGAGATCAACCCTGAGGAAACTGAAGCCTGAGAGTATGTACGAGACTTGGGAGGATGACCTGGATGGAATCCACACTGTCGCCTTTGCAGAGGAAGCAGATCCTGATGGCTATGAGTTCTTAGAGACTCTCAAGGCTGTGGCCCAAGACAACACTGAGAACCCCGACCTCAGTATCATCTGGATTGATCCTGATGACTTCCCGCTGCTGGTCCCGTACTGGGAGAAGACCTTTGACATTGACCTGTCAGCTCCACAAATAGGAGTTGTCAATGTTACAGACGCGGACAGCATATGGATGGAGATGGATAACGAGGAGGACCTGCCTTCTGCTGATGAGCTGGAGGACTGGCTGGAGGACGTGCTGGAGGGCGAGATCAACACAGAGGATGACGACGACGATGACGACGATGACGATGATGACGATGATGACGACGACGGATCCGGGCCCTCTAGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACGAGCACAACGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGGACACCGAATCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGGAGGTGGAGGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGT <SEQ ID No.: 116; PRT;Artificial sequence>ITCSLASVLKLMGARAVSELRLALLFVLVLGTPRLGVQGEDGLDFPEYDGVDRVINVNAKNYKNVFKKYEVLALLYHEPPEDDKASQRQFEMEELILELAAQVLEDKGVGFGLVDSEKDAAVAKKLGLTEEDSVYVFKGDEVIEYDGEFSADTLVEFLLDVLEDPVELIEGERELQAFENIEDEIKLIGYFKSKDSEHYKAYEDAAEEFHPYIPFFATFDSKVAKKLTLKLNEIDFYEAFMEEPMTIPDKPNSEEEIVSFVEEHRRSTLRKLKPESMYETVVEDDLDGIHTVAFAEEADPDGYEFLETLKAVAQDNTENPDLSIIWIDPDDFPLLVPYWEKTFDIDLSAPQIGVVNVTDADSIWMEMDNEEDLPSADELEDWLEDVLEGEINTEDDDDDDDDDDDDDDDDDGSGPSRMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNEHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLDTESALSKDPNEKRDHMVLLEEVEAAGITLGMDELYK-EFXXQISSTVAAARV-RARLNPLISLDCAF-LPAICC <SEQ ID No.: 117;DNA; Artificial sequence>TATTACGTGTTCGCTGGCTaGCGTTTaACTTaAGCTTATGGGGGCCAGAGCAGTGTCCGAGCTGCGGCTGGCACTGCTGTTTGTACTGGTGCTAGGGACGCCCAGGTTAGGGGTCCAGGGGGAAGATGGGTTGGACTTCCCTGAGTACGACGGTGTGGACCGTGTGATCAATGTGAATGCCAAGAACTACAAGAACGTGTTTAAGAAGTATGAGGTGCTGGCCCTCCTCTACCATGAGCCCCCTGAGGACGACAAGGCCTCGCAGAGACAATTTGAGATGGAGGAGCTAATCCTGGAGTTAGCAGCCCAAGTCTTAGAAGACAAGGGTGTTGGCTTTGGCCTGGTGGACTCAGAGAAGGATGCAGCTGTGGCCAAGAAACTAGGACTAACTGAAGAAGACAGCGTTTATGTGTTCAAAGGAGATGAAGTCATTGAATATGACGGCGAGTTTTCTGCAGACACTCTGGTGGAGTTTCTGCTTGATGTCCTAGAAGACCCTGTAGAGTTGATTGAAGGTGAACGAGAGCTGCAGGCATTTGAGAATATTGAAGATGAAATCAAACTCATTGGCTACTTCAAGAGCAAAGACTCAGAGCATTACAAAGCCTACGAGGACGCAGCTGAAGAGTTCCATCCCTACATCCCTTTCTTCGCTACCTTCGACAGCAAGGTGGCAAAGAAGCTGACTCTGAAGTTGAATGAGATTGATTTCTACGAGGCCTTCATGGAAGAGCCTATGACCATCCCAGACAAGCCCAACAGTGAAGAGGAGATTGTGAGCTTCGTGGAGGAGCACAGGAGATCAACCCTGAGGAAACTGAAGCCTGAGAGTATGTACGAGACTTGGGAGGATGACCTGGATGGAATCCACACTGTCGCCTTTGCAGAGGAAGCAGATCCTGATGGCTATGAGTICTTAGAGACTCTCAAGGCTGTGGCCCAAGACAACACTGAGAACCCCGACCTCAGTATCATCTGGATTGATCCTGATGACTTCCCGCTGCTGGTCCCGTACTGGGAGAAGACCTTTGACATTGACCTGTCAGCTCCACAAATAGGAGTTGTCAATGTTACAGACGCGGACAGCATATGGATGGAGATGGATAACGAGGAGGACCTGCCTTCTGCTGATGAGCTGGAGGACTGGCTGGAGGACGTGCTGGAGGGCGAGATCAACACAGAGTGATGACGATGATGACGACGACGGATCCGGGCCCTCTAGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACGAGCACAACGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGGACACCGAATCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGGAGGTGGAGGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGT <SEQ ID No.: 118; PRT; Artificial sequence>ITCSLASVLKLMGARAVSELRLALLFVLVLGTPRLGVQGEDGLDFPEYDGVDRVINVNAKNYKNVFKKYEVLALLYHEPPEDDKASQRQFEMEELILELAAQVLEDKGVGFGLVDSEKDAAVAKKLGLTEEDSVYVFKGDEVIEYDGEFSADTLVEFLLDVLEDPVELIEGERELQAFENIEDEIKLIGYFKSKDSEHYKAYEDAAEEFHPYIPFFATFDSKVAKKLTLKLNEIDFYEAFMEEPMTIPDKPNSEEEIVSFVEEHRRSTLRKLKPESMYETWEDDLDGIHTVAFAEEADPDGYEFLETLKAVAQDNTENPDLSIIWIDPDDFPLLVPYWEKTFDIDLSAPQIGVVNVTDADSIWMEMDNEEDLPSADELEDWLEDVLEGEINTEGSGPSRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNEHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLDTESALSKDPNEKRDHMVLLEEVEAAGITLGMDELYKEFXXQISSTVAAARVRARLNPLISLDCAFLPAICC <SEQ ID No.: 119; PRT; Artificialsequence> FCLTLRRRYTMGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSGPSRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNEHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLDIESALSKDPNEKRDHMVLLEEVEAAGITLGMDELYKEFELRRQACGRTRVPPPPPLRSGCQSPKGSXGCCPTPLPXXRIRPXQRPXXSAXXXXCX <SEQ ID No.: 120; PRT; Artificialsequence>XITCSLASVLKLGTELGSGPSRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNEHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLDTESALSKDPNEKRDHMVLLEEVEAAGITLGMDELYKFYTLRFLALFLAFAINFILLFYKVSEFCRYPAQWRPLESRGPVTRSASTVPSSCQPSV <SEQ IDNo.: 121; PRT3; > (D8-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProlIeLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:122; PRT3; > (D9-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:123; PRT3; > (D10-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:124; PRT3; > (D11-EGFP, CatchER without ER tag)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:125; PRT3; > (D11-EGFP, CatchER with ER tag)MetLeuLeuSerValProLeuLeuLeuGlyLeuLeuGlyLeuAlaAlaAlaAspValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLysLysAspGluLeu <SEQ ID No.: 126;PRT3; > (D12-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluAspValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:127; PRT3; > (D13-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrAsnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluAspValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ IDNo.: 128; PRT3; > (D14-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisGluValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:129; PRT3; > (D15-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisGluValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:130; PRT3; > (D16-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisGluValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:131; PRT3; > (D11-EGFP-2031, CatchER-203I)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspIleGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:132; PRT3; > (D11-EGFP-203V, CatchER-203V)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspValGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:133; PRT3; > (D11-EGFP-203D, CatchER-203D)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspAspGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ IDNo.: 134; PRT3; > (D11-EGFP-203F, CatchER-203F)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspPheGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ IDNo.: 135; PRT3; > (D11-EGFP-203E, CatchER-203E)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspGluGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:136; PRT3; > (D11-EGFP-175G, CatchER-175G)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlyGlyValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.:137; PRT3; > (D11-EGFP-148D, CatchER-148D)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluAspAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ IDNo.: 138; PRT3; > (G1M1-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnAspIleGluLeuLysGlyIIeAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 139; PRT3; > (G1M2-EGFP)ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuGluGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No.: 140; PRT; > (calsequestrin tethered CatchER)ITCSLASVLKLMGARAVSELRLALLFVLVLGTPRLGVQGEDGLDFPEYDGVDRVINVNAKNYKNVFKKYEVLALLYHEPPEDDKASQRQFEMEELILELAAQVLEDKGVGFGLVDSEKDAAVAKKLGLTEEDSVYVFKGDEVIEYDGEFSADTLVEFLLDVLEDPVELIEGERELQAFENIEDEIKLIGYFKSKDSEHYKAYEDAAEEFHPYIPFFATFDSKVAKKLTLKLNEIDFYEAFMEEPMTIPDKPNSEEEIVSFVEEHRRSTLRKLKPESMYETVVEDDLDGIHTVAFAEEADPDGYEFLETLKAVAQDNTENPDLSIIWIDPDDFPLLVPYWEKTFDIDLSAPQIGVVNVTDADSIWMEMDNEEDLPSADELEDWLEDVLEGEINTEDDDDDDDDDDDDDDDDDGSGPSRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNEHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLDTESALSKDPNEKRDHMVLLEEVEAAGITLGMDELYK-EFXXQISSTVAAARV-RARLNPLISLDCAF-LPAICC <SEQ ID No.: 141;PRT; > (calsequestrin 17 Asp deleted tethered CatchER)ITCSLASVLKLMGARAVSELRLALLFVLVLGTPRLGVQGEDGLDFPEYDGVDRVINVNAKNYKNVFKKYEVLALLYHEPPEDDKASQRQFEMEELILELAAQVLEDKGVGFGLVDSEKDAAVAKKLGLTEEDSVYVFKGDEVIEYDGEFSADTLVEFLLDVLEDPVELIEGERELQAFENIEDEIKLIGYFKSKDSEHYKAYEDAAEEFHPYIPFFATFDSKVAKKLTLKLNEIDFYEAFMEEPMTIPDKPNSEEEIVSFVEEHRRSTLRKLKPESMYETVVEDDLDGIHTVAFAEEADPDGYEFLETLKAVAQDNTENPDLSIIWIDPDDFPLLVPYWEKTFDIDLSAPQIGVVNVTDADSIWMEMDNEEDLPSADELEDWLEDVLEGEINTEGSGPSRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNEHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLDTESALSKDPNEKRDHMVLLEEVEAAGITLGMDELYKEFXXQISSTVAAARVRARLNPLISLDCAFLPAICC <SEQ ID No. 142: D8-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.143: D9-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.144: D10-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.145: D11-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.146: D12-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrAraAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGIyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluAspValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.147: D13-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAsnPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrAsnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluAspValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.148: D14-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisGluValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.149: D15-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisGluValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.150: D16-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisGluValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.151: D11-EGFP-203I>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspIleGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.152: D11-EGFP-203V>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspValGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.153: D11-EGFP-203D>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspAspGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.154: D11-EGFP-203F>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspPheGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.155: D11-EGFP-203E>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspGluGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.156: D11-EGFP-175G>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluAspGlyGlyValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.157: D11-EGFP-148D>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnGluAspAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnsnPheLysIleArgHisAsnIleGluAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuAspThrGluSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluGluValGluAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys <SEQ ID No.158: G1M1-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnAspIleGluLeuLysGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys<SEQ ID No. 159: G1M2-EGFP>ValSerLysGlyGluGluLeuPheThrGlyValValProIleLeuValGluLeuAspGlyAspLeuAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCysThrThrGlyLysLeuProValProTrpProThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArgThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluValLysPheGluGlyAspThrLeuValAsnArgIleGluLeuGluGlyIleAspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsnTyrAsnSerHisAsnValTyrIleThrAlaAspLysGlnLysAsnGlyIleLysAlaAsnPheLysIleArgHisAsnIleGluGluGluGluIleArgGluAlaPheArgValPheAspLysAspGlyAsnGlyTyrIleSerAlaAlaGluLeuArgHisValMetThrAsnLeuAspGlySerValGlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyProValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSerLysAspProAsnGluLysArgAspHisIleValLeuLeuGluPheValThrAlaAlaGlyIleThrLeuGlyMetAspGluLeuTyrLys

1-41. (canceled)
 42. An analyte sensor comprising an engineeredfluorescent host polypeptide having a metal ion binding site comprisinga plurality of negatively-charged amino acid residues providing aplurality of oxygen atoms orientated in a pentagonal bipyrimdalgeometry, wherein said metal ion binding site interacts with achromophore region of the engineered fluorescent host polypeptide suchthat binding of a metal ion analyte to the metal ion binding sitemodulates the emission of a fluorescent signal emitted by thefluorescent host polypeptide or modulates the absorbance spectrum of theengineered fluorescent host polypeptide, and wherein thenegatively-charged amino acids are at positions 147, 202, 204, and 223,and optionally at positions 149 and 225, according to the amino acidsequence SEQ ID NO:
 105. 43. The analyte sensor of claim 42, wherein theanalyte sensor includes a targeting motif for selectively targeting anendoplasmic reticulum, a sarcoplasmic reticulum, or a mitochondrion of acell.
 44. The analyte sensor of claim 42, wherein the analyte sensorincludes a targeting motif for selectively targeting an endoplasmicreticulum of a cell.
 45. The analyte sensor of claim 42, wherein theanalyte sensor binds to a metal ion selected from the group consistingof: calcium, lead, gadolinium, lanthanum, terbium, antimony, strontium,mercury, and cadmium.
 46. The analyte sensor of claim 42, wherein theanalyte sensor in the absence of a metal ion analyte emits a firstfluorescent signal and in the presence of a metal ion analyte bound tothe analyte sensor emits a second fluorescent signal, wherein the firstand the second fluorescent signals are distinguishably detectable. 47.The analyte sensor of claim 42, wherein the negatively-charged aminoacid residues are on the surface of three anti-parallel beta-sheets oron three strands of the protein having a beta-can structure.
 48. Amethod of detecting a metal ion analyte, comprising: (i) providing ananalyte sensor according to claim 1; (ii) providing a test samplesuspected of comprising the metal ion analyte having affinity for themetal ion binding site of the analyte sensor; (iii) detecting a firstfluorescent signal emitted by the analyte sensor in the absence of atest sample suspected of comprising said metal ion analyte; (iv)contacting the analyte sensor with the test sample; (v) detecting asecond fluorescent signal emitted by the analyte sensor in contact withthe test sample; and (vi) comparing the first fluorescent signal and thesecond fluorescent signal, wherein a ratiometric change in the signalindicates a metal ion analyte in the test sample is interacting with theanalyte sensor.
 49. The method of claim 48, wherein if the firstfluorescent signal and the second fluorescent signal differ in theirwavelengths, in their intensities, or in both their wavelengths andtheir intensities, an interaction between said metal ion analyte and theanalyte sensor is indicated.
 50. The method of claim 48, furthercomprising the step of determining from the ratiometric change aquantitative measurement of the metal ion analyte in the test sample.51. The method of claim 48, wherein the metal ion analyte is selectedfrom the group consisting of: calcium, lead, gadolinium, lanthanum,terbium, antimony, strontium, mercury, and cadmium.
 52. A recombinantnucleic acid encoding an analyte sensor according to claim
 42. 53. Therecombinant nucleic acid of claim 52, further comprising a vectornucleic acid sequence.
 54. The recombinant nucleic acid of claim 52,wherein the recombinant nucleic acid is in a genetically modified cell.55. A method for characterizing the cellular activity of a metal ionanalyte comprising: (i) providing a genetically-modified cell expressingan analyte sensor according to claim 42; (ii) detecting a firstfluorescent signal emitted by the analyte sensor; (iii) detecting asecond fluorescent signal emitted by the analyte sensor after theinduction of a physiological event in the cell; and (iv) comparing thefirst and the second fluorescent signals wherein, if the first and thesecond fluorescent signals differ in at least one of their wavelengthsor their intensities, a change in the level of the analyte in the cellinduced by the physiological event in said cell is indicated, andwherein the metal ion analyte is calcium, lead, gadolinium, lanthanum,terbium, antimony, strontium, mercury, or cadmium.
 56. A method ofdetecting a metal ion analyte, comprising: (i) providing an analytesensor according to claim 1; (ii) providing a test sample suspected ofcomprising a metal ion analyte having affinity for the metal ion bindingsite of the analyte sensor; (iii) detecting either (a) a firstabsorption signal derived from the analyte sensor in the absence of athe test sample or (b) a first fluorescent signal emitted by the analytesensor in the absence of the test sample; (iv) contacting the analytesensor with the test sample; (v) detecting either (a) a secondabsorption signal derived from the analyte sensor in contact with thetest sample, or (b) a second fluorescent signal emitted by the analytesensor in contact with the test sample; and (vi) comparing either (a)the first and the second absorption signals, wherein a ratiometricchange in the absorption signal indicates said metal ion analyte in thetest sample is interacting with the analyte sensor, or (b) the first andthe second fluorescent signal, wherein a ratiometric change in thelifetime of the signal indicates said metal ion analyte in the testsample is interacting with the analyte sensor.